Stand. Genomic Sci. 2014 9:3
doi:10.4056/sigs.5281010
The complete genome sequence of Clostridium indolis DSM 755T

Amy S. Biddle1,2, Susan Leschine3, Marcel Huntemann4, James Han4, Amy Chen4, Nikos Kyrpides4, Victor Markowitz4, Krishna Palaniappan4, Natalia Ivanova4, Natalia Mikhailova4, Galina Ovchinnikova4, Andrew Schaumberg4, Amrita Pati4, Dimitrios Stamatis4, Tatiparthi Reddy4, Elizabeth Lobos4, Lynne Goodwin4, Henrik P. Nordberg4, Michael N. Cantor4, Susan X. Hua4, Tanja Woyke4, Jeffrey L. Blanchard2,5,6

1 Department of Microbiology, University of Massachusetts, Amherst, MA, USA
2Institute for Cellular Engineering, University of Massachusetts, Amherst, MA, USA
3Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA
4 Joint Genome Institute, Walnut Creek, CA, USA
5 Department of Biology, University of Massachusetts, Amherst, MA, USA
6 Graduate Program in Organismal and Evolutionary Biology, University of Massachusetts, Amherst, MA, USA.

Correspondence: Amy S. Biddle (abiddle@microbio.umass.edu)

epub-ppub publication date: March 18, 2014.

Abstract

Clostridium indolis DSM 755T is a bacterium commonly found in soils and the feces of birds and mammals. Despite its prevalence, little is known about the ecology or physiology of this species. However, close relatives, C. saccharolyticum and C. hathewayi, have demonstrated interesting metabolic potentials related to plant degradation and human health. The genome of C. indolis DSM 755T reveals an abundance of genes in functional groups associated with the transport and utilization of carbohydrates, as well as citrate, lactate, and aromatics. Ecologically relevant gene clusters related to nitrogen fixation and a unique type of bacterial microcompartment, the CoAT BMC, are also detected. Our genome analysis suggests hypotheses to be tested in future culture based work to better understand the physiology of this poorly described species.

Keywords: Clostridium indolis, citrate, lactate, aromatic degradation, nitrogen fixation, bacterial microcompartments.

Copyright © retained by original authors
Introduction

The C. saccharolyticum species group is a poorly described and taxonomically confusing clade in the Lachnospiraceae, a family within the Clostridiales that includes members of clostridial cluster XIVa [1]. This group includes C. indolis, C. sphenoides, C. methoxybenzovorans, C. celerecrescens, and Desulfotomaculum guttoideum, none of which are well studied (Figure 1). C. saccharolyticum has gained attention because its saccharolytic capacity was shown to be syntrophic with the cellulolytic activity of Bacteroides cellulosolvens in co-culture, enabling the conversion of cellulose to ethanol in a single step [6,7]. Members of this group, such as C. celerecrescens, are themselves cellulolytic [8], and others are known to degrade unusual substrates such as methylated aromatic compounds (C. methoxybenzovorans) [9], and the insecticide lindane (C. sphenoides) [10]. C. indolis was targeted for whole genome sequencing to provide insight into the genetic potential of this taxa that could then direct experimental efforts to understand its physiology and ecology.

Figure 1
Figure 1
Figure 1

Phylogenetic tree based on 16S rRNA gene sequences highlighting the position of Clostridium indolis relative to other type strains (T) within the Lachnospiraceae. The strains and their corresponding NCBI accession numbers (and, when applicable, draft sequence coordinates) for 16S rRNA genes are: Desulfotomaculum guttoideum strain DSM 4024T, Y11568; C. sphenoides ATCC 19403T, AB075772; C. celerecrescens DSM 5628T, X71848; C. indolis DSM 755T, Pending release by JGI: 1620643-1622056; C. methoxybenzovorans SR3, AF067965; C. saccharolyticum WM1T, NC_014376:18567-20085; C. algidixylanolyticum SPL73T, AF092549; C. hathewayi DSM 13479T, ADLN00000000: 202-1639; Eubacterium eligens L34420 T, L34420; Ruminococcus gnavus ATCC 29149T, X94967; R. torques ATCC 27756T, L76604; E. rectale L34627T; Roseburia intestinalis L1-82T, AJ312385; R. hominis A2-183T, AJ270482; C. jejuense HY-35-12T, AY494606; C. xylanovorans HESP1T, AF116920; C. phytofermentans ISDgT, CP000885: 15754-17276. The tree uses sequences aligned by MUSCLE, and was inferred using the Neighbor-Joining method [2]. The optimal tree with the sum of branch lengths = 0.50791241 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [3]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [4] and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA 5 [5]. C. stercorarium ATCC 35414T, CP003992: 856992-858513 was used as an outgroup.


Classification and features

The general features of Clostridium indolis DSM 755T are listed in Table 1. C. indolis DSM 755T was originally named for its ability to hydrolyze tryptophan to indole, pyruvate, and ammonia [23] in the classic Indole Test used to distinguish bacterial species. It has been isolated from soil [24], feces [25], and clinical samples from infections [27]. Despite its prevalence, C. indolis is not well characterized, and there are conflicting reports about its physiology. It is described as a sulfate reducer with the ability to ferment some simple sugars, pectin, pectate, mannitol, and galacturonate, and convert pyruvate to acetate, formate, ethanol, and butyrate [28]. According to this source, neither lactate nor citrate are utilized, however other studies demonstrate that fecal isolates closely related to C. indolis may utilize lactate [29], and that the type strain DSM 755T utilizes citrate [30]. It is unclear whether C. indolis is able to make use of a wider range of sugars or break down complex carbohydrates, however growth is reported to be stimulated by fermentable carbohydrates [28].

Table 1: Classification and general features of Clostridium indolis DSM 755T
MIGS ID      Property     Term     Evidence Code
    Domain Bacteria     TAS [11]
    Phylum Firmicutes     TAS [12-14]
    Class Clostridia     TAS [15,16]
     Current classification     Order Clostridiales     TAS [17,18]
    Family Lachnospiraceae     TAS [15,19]
    Genus Clostridium     TAS [17,20,21]
    Species Clostridium indolis     TAS [17,22]
    Type strain DSM 755
     Gram stain     Negative     TAS [23,24]
     Cell shape     Rod     TAS [23,24]
     Motility     Motile     TAS [23,24]
     Sporulation     Terminal, spherical spores     TAS [23,24]
     Temperature range     Mesophilic     TAS [23,24]
     Optimum temperature     37oC     TAS [23,24]
     Carbon sources     Glucose, lactose, sucrose, mannitol, pectin, pyruvate, others     TAS [23,24]
     Terminal electron receptor     Sulfate     TAS [23,24]
     Indole test     Positive     TAS [23,24]
MIGS-6      Habitat     Isolated from soil, feces, wounds     TAS [24,25]
MIGS-6.3      Salinity     Inhibited by 6.5% NaCl     TAS [23,24]
MIGS-22      Oxygen     Anaerobic     TAS [23,24]
MIGS-15      Biotic relationship     Free living and host associated TAS [24,25],9
MIGS-14      Pathogenicity     No NAS
MIGS-4      Geographic location     Soil, feces TAS [24,25],9

Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [26].

Genome sequencing information
Genome project history

The genome was selected based on the relatedness of C. indolis DSM 755T to C. saccharolyticum, an organism with interesting saccharolytic and syntrophic properties. The genome sequence was completed on May 2, 2013, and presented for public access on June 3, 2013. Quality assurance and annotation done by DOE Joint Genome Institute (JGI) as described below. Table 2 presents a summary of the project information and its association with MIGS version 2.0 compliance [31].

Table 2: Project information
MIGS ID     Property     Term
MIGS-31     Finishing quality     Improved Draft
MIGS-28     Libraries used     Shotgun and long insert mate pair (Illumina), SMRTbellTM (PacBio)
MIGS-29     Sequencing platforms     Illumina and PacBio
MIGS-31.2     Fold coverage     759.7× (Illumina), 51.6× (PacBio)
MIGS-30     Assemblers     Velvet, AllpathsLG
MIGS-32     Gene calling method     Prodigal, GenePRIMP
    Genome Database release     June 3, 2013 (IMB)
    Genbank ID     Pending release by JGI
    Genbank Date of Release     Pending release by JGI
    GOLD ID     Gi22434
    Project relevance     Anaerobic plant degradation
Growth conditions and DNA isolation

C. indolis DSM 755T was cultivated anaerobically on GS2 medium as described elsewhere [32]. DNA for sequencing was extracted using the DNA Isolation Bacterial Protocol available through the JGI (http://www.jgi.doe.gov). The quality of DNA extracted was assessed by gel electrophoresis and NanoDrop (ThermoScientific, Wilmington, DE) according to the JGI recommendations, and the quantity was measured using the Quant-iTTM Picogreen assay kit (Invitrogen, Carlsbad, CA) as directed.

Genome sequencing and assembly

The draft genome of C. indolis was generated at the DOE Joint genome Institute (JGI) using a hybrid of the Illumina and Pacific Biosciences (PacBio) technologies. An Illumina std shotgun library and long insert mate pair library was constructed and sequenced using the Illumina HiSeq 2000 platform [33]. 16,165,490 reads totaling 2,424.8 Mb were generated from the std shotgun and 26,787,478 reads totaling 2,437.7 Mb were generated from the long insert mate pair library. A Pacbio SMRTbellTM library was constructed and sequenced on the PacBio RS platform. 99,448 raw PacBio reads yielded 118,743 adapter trimmed and quality filtered subreads totaling 330.2 Mb. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts [34]. Filtered Illumina and PacBio reads were assembled using AllpathsLG (PrepareAllpathsInputs: PHRED 64=1 PLOIDY=1 FRAG COVERAGE=50 JUMP COVERAGE=25; RunAllpath- sLG: THREADS=8 RUN=std pairs TARGETS=standard VAPI WARN ONLY=True OVERWRITE=True) [35]. The final draft assembly contained 1 contig in 1 scaffold. The total size of the genome is 6.4 Mb. The final assembly is based on 2,424.6 Mb of Illumina Std PE, 2,437.6 Mb of Illumina CLIP PE and 330.2 Mb of PacBio post filtered data, which provides an average 759.7× Illumina coverage and 51.6× PacBio coverage of the genome, respectively.

Genome annotation

Genes were identified using Prodigal [36], followed by a round of manual curation using GenePRIMP [9] for finished genomes and Draft genomes in fewer than 10 scaffolds. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool [37] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [38]. Other non–coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL [39]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform [40] developed by the Joint Genome Institute, Walnut Creek, CA, USA [41]. Information in the tables below reflects the gene information in the JGI annotation on the IMG website [40].

Genome properties

The genome of C. indolis DSM 755 consists of a 6,383,701 bp circular chromosome with GC content of 44.93% (Table 3). Of the 5,903 genes predicted, 5,802 were protein-coding genes, and 101 RNAs; 170 pseudogenes were also identified. 81.21% of genes were assigned with a putative function with the remaining annotated as hypothetical proteins. The genome summary and distribution of genes into COGs functional categories are listed in Tables 3 and 4.

Table 3: Nucleotide content and gene count levels of the genome of C. indolis DSM 755
Attribute      Value     % of totala
Genome size (bp)      6,383,701
DNA Coding region (bp)      5,688,007     89.10
DNA G+C content (bp)      2,868,247     44.93
Total genesb      5,903     100.00
RNA genes      101     1.71
Protein-coding genes      5,802     98.29
Protein-coding with function pred.      4,794     81.21
Genes in paralog clusters      4,527     76.69
Genes assigned to COGs      4,643     78.65
Genes with signal peptides      421     7.13
Genes with transmembrane helices      1,494     25.31
Paralogous groups      4,527     76.69

a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

b) Also includes 170 pseudogenes.

Table 4: Number of genes in C. indolis DSM 755 associated with the 25 general COG functional categories
Code     Value     %agea      Description
J     184     3.57      Translation
A     0     0      RNA processing and modification
K     531     10.30      Transcription
L     191     3.71      Replication, recombination and repair
B     1     0.02      Chromatin structure and dynamics
D     28     0.54      Cell cycle control, mitosis and meiosis
Y     0     0      Nuclear structure
V     107     2.08      Defense mechanisms
T     335     6.50      Signal transduction mechanisms
M     235     4.56      Cell wall/membrane biogenesis
N     70     1.36      Cell motility
Z     0     0      Cytoskeleton
W     0     0      Extracellular structures
U     41     0.80      Intracellular trafficking and secretion
O     124     2.41      Posttranslational modification, protein turnover, chaperones
C     261     5.06      Energy production and conversion
G     910     17.65      Carbohydrate transport and metabolism
E     493     9.56      Amino acid transport and metabolism
F     110     2.13      Nucleotide transport and metabolism
H     153     2.97      Coenzyme transport and metabolism
I     77     1.49      Lipid transport and metabolism
P     325     6.30      Inorganic ion transport and metabolism
Q     70     1.36      Secondary metabolites biosynthesis, transport and catabolism
R     590     11.45      General function prediction only
S     319     6.19      Function unknown
-     1260     21.35      Not in COGs

a) The total is based on the total number of protein coding genes in the annotated genome.

The genomes of C. indolis and its near relatives (C. saccharolyticum, C. hathewayi, and C. phytofermentans) have similar numbers of genes in each of the 25 broad COG categories (not shown), however differences exist in the type and distribution of genes in specific functional groups (Table 5), particularly those related to COG categories (G) Carbohydrate transport and metabolism, (C) Energy production and conversion, and (Q) Secondary metabolites biosynthesis, transport and catabolism.

Table 5: Number of genes in each of the 25 general COG functional categoriesa found in C. indolis DSM 755T but not in closely related species
Code     Value      Description
J     4      Translation
A     0      RNA processing and modification
K     5      Transcription
L     9      Replication, recombination and repair
B     1      Chromatin structure and dynamics
D     0      Cell cycle control, mitosis and meiosis
Y     0      Nuclear structure
V     1      Defense mechanisms
T     2      Signal transduction mechanisms
M     8      Cell wall/membrane biogenesis
N     2      Cell motility
Z     0      Cytoskeleton
W     0      Extracellular structures
U     1      Intracellular trafficking and secretion
O     10      Posttranslational modification, protein turnover, chaperones
C     28      Energy production and conversion
G     6      Carbohydrate transport and metabolism
E     8      Amino acid transport and metabolism
F     1      Nucleotide transport and metabolism
H     11      Coenzyme transport and metabolism
I     2      Lipid transport and metabolism
P     11      Inorganic ion transport and metabolism
Q     10      Secondary metabolites biosynthesis, transport and catabolism
R     18      General function prediction only
S     21      Function unknown

a) Number of genes from a set of 158 genes not found in near relatives (C. saccharolyticum, C. phytofermentans, C. hathewayi) associated with the 25 general COG functional categories.

Carbohydrate transport and metabolism

Plant biomass is a complex composite of fibrils and sheets of cellulose, hemicellulose, waxes, pectin, proteins, and lignin. Bacteria from soil and the gut generally possess a variety of genes to degrade and transport the diversity of substrates encountered in these plant-rich environments. The genome of C. indolis includes 910 genes (17.65% of total protein coding genes) in this COG group including glycoside hydrolases with the potential to degrade complex carbohydrates including starch, cellulose, and chitin (Table 6), as well as an abundance of carbohydrate transporters (Figure 2).

Table 6: Selected carbohydrate active genes in the C. indolis DSM 755T genome
Gene count     Product namea      Database IDb
19     Beta-glucosidase (GH-1)      EC:3.2.1.86
8     Beta-galactosidase/
    beta-glucuronidase (GH-2)
     EC:3.2.1.23
     EC:3.2.1.25
     EC:3.2.1.31
7     Beta-glucosidase/ related glucosidases (GH-3)      EC:3.2.1.21
     EC:3.2.1.52
14     Alpha-galactosidases/
    6-phospho-beta-glucosidases (GH-4)
     EC:3.2.1.86
     EC:3.2.1.122
     EC:3.2.1.22
2     Cellulase, endogluconase (GH-5)      EC:3.2.1.4
14     Alpha-amylase      EC:3.2.1.10
     EC:3.2.1.20
     EC:2.4.1.7
     EC:3.2.1.70
8     Beta-xylosidase (GH 39)      EC:3.2.1.37
2     Chitinase (GH 18)      EC:3.2.1.14

a) GH designations given from the CAZy database [42]. b) Enzyme Commission (EC) numbers assigned by the Integrated Microbial Genome (IMG) database [41].

Figure 2
Figure 2
Figure 2

Distribution of ABC and PTS transporters in the genomes of C. indolis and related genomes determined from Integrated Microbial Genome (IMG) annotation [40] viewed based on (a) Total umber of COGS, and (b) Percentage of genes in the genome.


Almost 8% of the protein-coding genes in the genome of C. indolis were found to be associated with carbohydrate transport, represented by two main strategies. ABC (ATP binding cassette) transporters tend to carry oligosaccharides, and have less affinity for hexoses [43,44], while PTS (phosphotransferase system) transporters carry many different mono- and disaccharides, especially hexoses [45]. PTS systems provide a means of regulation via catabolite repression [46], and are thought to enable bacteria living in carbohydrate-limited environments to more efficiently utilize and compete for substrates [46]. Both C. indolis and its near relatives are more highly enriched in ABC than PTS transporters (Fig 2), however nearly a third of C. indolis and C. saccharolyticum transporters are PTS genes, suggesting a preference for hexoses, as well as an adaptation to more marginal environments. C. indolis also possesses ten genes associated with all three components of the TRAP-type C4-dicarboxylate transport system, which transports C4-dicarboxylates such as formate, succinate, and malate [47], as well as six putative malate dehydrogenases and two putative succinate dehydrogenases suggesting that C. indolis may have the potential to utilize both of these short chain fatty acids.

Energy production and conversion

The genome of C. indolis contains 261 genes in COG category (C) Energy production and conversion, 28 of which are not found in the near relatives analyzed, including genes for citrate utilization (Table 7) and nitrogen fixation (Table 8).

Table 7: Selection of C. indolis DSM 755 genes related to citrate utilization.
Locus Tag      Putative Gene Producta     Gene IDa
K401DRAFT_2892      holo-ACP synthase (CitX)     EC:2.7.7.61
K401DRAFT_2893      citrate lyase acyl carrier (CitD)     EC:4.1.3.6
K401DRAFT_2894      citrate lyase beta subunit (CitE)     EC:4.1.3.6
    EC:2.8.3.10
K401DRAFT_2895      citrate lyase alpha subunit (CitF)     EC:4.1.3.6
    EC:2.8.3.10
K401DRAFT_2896      triphosphoribosyl-dephospho-CoA synthase (CitG)     EC:2.7.8.25
K401DRAFT_2897      citrate (pro3S)-lyase ligase (CitC)     EC:6.2.1.22
K401DRAFT_2898      response regulator, CheY-like receiver domain, winged helix DNA binding domain     -
K401DRAFT_2899      signal transduction histidine kinase     -
K401DRAFT_2900      citrate transporter, CITMHS family     KO:K03303
    TC.LCTP

Gene products and Enzyme Commission (EC) numbers assigned by the Integrated Microbial Genome (IMG) database [41].

Table 8: Selection of C. indolis DSM 755 genes related to nitrogen fixation.
Locus Tag     Putative Gene Product     Gene ID
K401DRAFT_0533     nitrogenase Mo-Fe protein, α and β chains     pfam00148
K401DRAFT_0534     nitrogenase Mo-Fe protein, α and β chains     pfam00148
K401DRAFT_0535     nitrogenase subunit (ATPase) (nifH)     pfam00142
K401DRAFT_0884     nitrogenase Mo-Fe protein, α and β chains     pfam00148
K401DRAFT_0885     nitrogenase Mo-Fe protein, α and β chains     pfam00148
K401DRAFT_0886     nitrogenase subunit (ATPase) (nifH)     pfam00142
K401DRAFT_3349     nitrogenase Mo-Fe protein, α and β chains     pfam00148
K401DRAFT_3350     nitrogenase Mo-Fe protein, α and β chains     pfam00148
K401DRAFT_3351     nitrogenase subunit (ATPase) (nifH)     pfam00142
K401DRAFT_3874     nitrogenase Mo-Fe protein, α and β chains (nifD)     pfam00148
K401DRAFT_3875     nitrogenase Mo-Fe protein, α and β chains (nifK)     pfam00148
K401DRAFT_3876     nitrogenase Fe protein     pfam00142
K401DRAFT_3878     nitrogenase Mo-Fe protein, α and β chains (nifD)     pfam00148
K401DRAFT_3879     nitrogenase Mo-Fe protein, α and β chains (nifK)     pfam00148
K401DRAFT_3880     dinitrogenase Fe-Mo cofactor, (nifH)     pfam02579
K401DRAFT_3895     nitrogenase Mo-Fe protein, α and β chains (nifD)     pfam00148
K401DRAFT_3896     nitrogenase Mo-Fe protein, α and β chains (nifK)     pfam00148
K401DRAFT_5519     nitrogenase Mo-Fe protein, α and β chains (nifB)     pfam04055
K401DRAFT_5520     nitrogenase Mo-Fe protein, α and β chains (nifE)     pfam00148
K401DRAFT_5521     nitrogenase Mo-Fe protein (nifK)     pfam00148
K401DRAFT_5522     nitrogenase component 1, alpha chain (nifN-like)     pfam00148
K401DRAFT_5525     nitrogenase subunit (ATPase) (nifH)     pfam00142

Nitrogenase genes have a common gene identifier (EC:1.18.6.1), therefore the pfam numbers are given to distinguish between subunits. Gene product names and pfam numbers assigned by the Integrated Microbial Genome (IMG) database [41].

Citrate utilization

Citrate is a metabolic intermediary found in all living cells. In aerobic bacteria, citrate is utilized as part of the tricarboxylic acid (TCA) cycle. In anaerobes, citrate is fermented to acetate, formate, and/or succinate. The first step is the conversion of citrate to acetate and oxaloacetate in a reaction catalyzed by citrate lyase (EC:4.1.3.6) [48]. C. sphenoides, a close relative of C. indolis that does not yet have a sequenced genome has been shown to utilize citrate [49], but there is conflicting evidence as to whether this phenotype is present in C. indolis [28,30]. The genome of C. indolis reveals a group of seven citrate genes organized in a cluster similar to operons found in other bacterial species [48,50] (Figure 3) including CitD, CitE, and CitF, the three subunits of the citrate lyase gene [48], CitG and CitX which have been shown to be necessary for citrate lyase function [50], CitMHS, a citrate transporter, and a putative two component system similar to citrate regulatory mechanisms in other bacteria [51].

Figure 3
Figure 3
Figure 3

Citrate utilization genes are in a single gene cluster on K401DRAFT_scaffold0000.1.1, including the citrate transporter CitMHS, and a putative two-component system.


Nitrogen Fixation

Nitrogen fixation has been observed in other clostridia [52,53] but has not been demonstrated in the C. saccharolyticum species group. It has been suggested that the capacity to fix nitrogen confers a selective advantage to cellulolytic microbes that live in nitrogen limited environments such as many soils [52]. The functional summary suggests that C. indolis can fix nitrogen. The C. indolis genome reveals 22 nitrogenase related genes in four gene clusters (Table 8), none of which are found in the near relatives analyzed in this study. A minimum set of six genes encoding for structural and biosynthetic components of a functional nitrogenase complex have been hypothesized [54]. Genes needed for the nitrogenase structural component proteins (nifH, nifD, and nifK) are present in C. indolis, but one of the three genes required to synthesize the nitrogenase iron-molybdenum cofactor (nifN) is not identified. Follow up experiments are needed to determine whether C. indolis can fix nitrogen as predicted by the genome analysis.

Lactate utilization

The genome of C. indolis includes both D- and L-lactate dehydrogenases, which convert lactate to pyruvate. Additionally, there is a lactate transporter, suggesting that C. indolis is able to utilize exogenous lactate [Table 9].

Table 9: Selection of C. indolis DSM 755 genes related to lactate utilization.
Locus Tag     Putative Gene Product     Gene ID
K401DRAFT_1877     L-lactate dehydrogenase     EC:1.1.1.27
K401DRAFT_5775     L-lactate dehydrogenase     EC:1.1.1.27
K401DRAFT_3431     L-lactate transporter, LctP family     TC.LCTP
K401DRAFT_3220     D-lactate dehydrogenase     EC:1.1.1.28

Annotations assigned by the Integrated Microbial Genome (IMG) database [41]

Bacterial microcompartments (BMC)

The C. indolis genome contains genes associated with bacterial microcompartment shell proteins. Bacterial microcompartments (BMCs) are proteinaceous organelles involved in the metabolism of ethanolamine, 1,2-propanediol, and possibly other metabolites (Rev in [55-57]). BMCs are often encoded by a single operon or contiguous stretch of DNA. The different metabolic types of BMCs can be distinguished by a key enzyme (e.g., ethanolamine lyase and propanediol dehydratase) related to its metabolic function. While the other associated genes in the operon can vary, they frequently include an alcohol dehydrogenase, an aldehyde dehydrogenase, an aldolase and an oxidoreductase.

In C. indolis there are 2 separate genetic loci that code for BMCs (Table 10 and 11 and Figure 4). One C. indolis locus (Table 10) contains a gene (K401DRAFT_2189) with sequence similarity to a B12-independent propanediol dehydratase found in Roseburia inulinivorans and Clostridium phytofermentans [58,59] (both members of the Lachnospiraceae). This enzyme has been shown to be involved in the metabolism of fucose and rhamnose [58,59] and was subsequently categorized as the glycyl radical prosthetic group-based (grp) BMC [60]. The glycyl radical family of enzymes was recently expanded to include a choline trimethylamine lyase activity that is part of a microcompartment loci in Desulfovibrio desulfuricans [61]. The corresponding C. indolis enzymes (K401DRAFT_2189 and K401DRAFT_2190) are more similar to the D. desulfuricans protein, but there are differences in the gene content of the microcompartment loci. Further work is needed to determine the physiological role of this microcompartment.

Table 10: grp-BMC genes found in the C. indolis genome.
Locus Tag       Product Name     Gene ID/ Protein Information
K401DRAFT_2181       Predicted transcriptional regulator     COG0789
K401DRAFT_2182       Predicted membrane protein     COG2510
K401DRAFT_2183       Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_2184       Predicted membrane protein     pfam00936
K401DRAFT_2185       Hypothetical protein     -
K401DRAFT_2186       Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_2187       Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_2188       NAD-dependent aldehyde dehydrogenase     pfam00171
K401DRAFT_2189       Pyruvate formate lyase     pfam02901
K401DRAFT_2190       Pyruvate formate lyase activating enzyme     pfam04055
K401DRAFT_2191       Ethanolamine utilization protein     pfam00936
K401DRAFT_2192       Ethanolamine utilization protein     pfam10662
K401DRAFT_2193       Alcohol dehydrogenase, class IV     pfam00465
K401DRAFT_2194       Ethanolamine utilization cobalamin adenosyltransferase     COG4892
K401DRAFT_2195       Ethanolamine utilization protein, possible chaperonin     COG4820
K401DRAFT_2196       Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_2197       Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam03319
K401DRAFT_2198       Ethanolamine utilization protein     pfam06249
K401DRAFT_2199       Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_2200       NAD-dependent aldehyde dehydrogenase     pfam00171
K401DRAFT_2201       Propanediol utilization protein     pfam06130
K401DRAFT_2202       Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936

Annotations assigned by the Integrated Microbial Genome (IMG) database [41].

Table 11: CoAT BMC genes found in the C. indolis genome.
Locus Tag      Product Name     Gene ID/ Protein Information
K401DRAFT_4970      DeoRC transcriptional regulator     pfam00455
K401DRAFT_4969      fucA, L-fuculose-phosphate aldolase     EC:4.1.2.17
K401DRAFT_4968      pduP, propionaldehyde dehydrogenase     pfam00171
K401DRAFT_4967      eutM, ethanolamine utilization protein     pfam00936
K401DRAFT_4966      Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_4965      Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_4964      Carbon dioxide concentrating mechanism/carboxysome shell protein     pfam00936
K401DRAFT_4963      Pdul, propanediol utilization protein     pfam06130
K401DRAFT_4962      eutN_CcmL     pfam03319
K401DRAFT_4961      SBP_bac_8, ABC-type sugar transporter     pfam13416
K401DRAFT_4960      Uncharacterized NAD(FAD)-dependent dehydrogenase     COG0446
K401DRAFT_4959      CoA-transferase     pfam01144
K401DRAFT_4958      CoA-transferase     pfam01144
K401DRAFT_4957      Fe-ADH, Alcohol dehydrogenase     pfam00465

Annotations assigned by the Integrated Microbial Genome (IMG) database [41]

Figure 4
Figure 4
Figure 4

CoAT BMC operon found in C. indolis, Caldalkalibacillus thermarum, C. stricklandii, C. saccharolyticum, and Bacillus selenitrireducens. Gene details are found in Table 11.


The second C. indolis BMC loci (Table 11 and Figure 4) is even more enigmatic. This loci contains the shell proteins, alcohol dehydrogenase, aldehyde dehydrogenase, aldolase and oxidoreductase commonly found in microcompartments, but it lacks a known key enzyme. Homologs of this operon were found in four other bacterial species (Figure 4). They are all missing a known key enzyme and contain 2 genes annotated as CoA-transferase. We propose that the C. indolis genome and these other bacteria contain a novel type of microcompartment, designated the CoAT BMC. It is not clear that the function of the 2 annotated CoA-transferase genes are as predicted and further research is needed to demonstrate the physiological role of this BMC.

Secondary metabolites biosynthesis, transport and catabolism

Protocatechuate and other aromatics are intermediaries in the degradation of lignin in plant rich environments [62]. The genome of C. indolis contains two protocatechuate dioxygenases and an aromatic hydrolase, revealing the potential for utilizing aromatic compounds (Table 12).

Table 12: Selection of C. indolis DSM 755T genes related to degradation of aromatics.
Locus Tag       Putative Gene Product     Gene ID
K401DRAFT_3571       Protocatechuate 3,4-dioxygenase beta subunit     EC:1.13.11.3
K401DRAFT_3568       Protocatechuate 3,4-dioxygenase beta subunit     EC:1.13.11.3
K401DRAFT_3412       Aromatic ring hydroxylase     EC:5.3.3.3
    EC:4.2.1.120

Annotations assigned by the Integrated Microbial Genome (IMG) database [41]

Conclusion

The genomic sequence of C. indolis reported here reveals the metabolic potential of this organism to utilize a wide assortment of fermentable carbohydrates and intermediates including citrate, lactate, malate, succinate, and aromatics, and points to potential ecological roles in nitrogen fixation and ethanolamine utilization. Further culture-based characterization is necessary to confirm the metabolic activity suggested by this genomic analysis, and to expand the description of C. indolis.

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