Skip to content

Advertisement

Open Access

High quality draft genome sequences of Mycoplasma agassizii strains PS6T and 723 isolated from Gopherus tortoises with upper respiratory tract disease

Standards in Genomic Sciences201813:12

https://doi.org/10.1186/s40793-018-0315-1

Received: 16 November 2017

Accepted: 4 April 2018

Published: 27 April 2018

Abstract

Mycoplasma agassizii is one of the known causative agents of upper respiratory tract disease (URTD) in Mojave desert tortoises (Gopherus agassizii) and in gopher tortoises (Gopherus polyphemus). We sequenced the genomes of M. agassizii strains PS6T (ATCC 700616) and 723 (ATCC 700617) isolated from the upper respiratory tract of a Mojave desert tortoise and a gopher tortoise, respectively, both with signs of URTD. The PS6T genome assembly was organized in eight scaffolds, had a total length of 1,274,972 bp, a G + C content of 28.43%, and contained 979 protein-coding genes, 13 pseudogenes and 35 RNA genes. The 723 genome assembly was organized in 40 scaffolds, had a total length of 1,211,209 bp, a G + C content of 28.34%, and contained 955 protein-coding genes, seven pseudogenes, and 35 RNA genes. Both genomes exhibit a very similar organization and very similar numbers of genes in each functional category. Pairs of orthologous genes encode proteins that are 93.57% identical on average. Homology searches identified a putative cytadhesin. These genomes will enable studies that will help understand the molecular bases of pathogenicity of this and other Mycoplasma species.

Keywords

Mycoplasma agassizii Desert tortoiseGopher tortoise Gopherus Upper respiratory tract disease (URTD)PS6T ATCC 700616723ATCC 700617

Introduction

The genus Mycoplasma , within the bacterial class Mollicutes ( Tenericutes ), contains over one hundred species, many of which are pathogenic to vertebrates [1]. An upper respiratory tract disease has been implicated in population declines in Mojave Desert tortoises ( Gopherus agassizii ) found in the desert southwest of the United States and gopher tortoises ( Gopherus polyphemus ) inhabiting forests of the U.S. southeast [24]. Pathogens associated with this disease include two Mycoplasma , Mycoplasma agassizii and Mycoplasma testudineum [57]. Due to conservation concerns regarding URTD, this disease and its associated pathogens have become a topic of research interest, though our understanding of the biology and progression of URTD is lacking [8, 9]. In particular, disease in tortoises is found with varying levels of morbidity, and one hypothesis for this finding is that there is genetic variation of M. agassizii associated with varying levels of virulence [8]. To understand better the amount of genomic differentiation occurring between M. agassizii collected from different tortoise host species, and to identify markers associated with virulence, we sequenced the M. agassizii genome from two strains, PS6T and 723. This sequencing is part of a larger project to ultimately genetically detect variation in strains and their virulence from field-cultured samples.

Organism information

Classification and features

Mycoplasma agassizii has been isolated from multiple tortoise species, and was found to be pathogenic in Mojave Desert tortoises and gopher tortoises in North America, causing URTD [5, 6, 10]. In infected North American tortoises, M. agassizii is most often found in the nasal passages and choana, but can also be isolated from the trachea and lungs [10]. This microbe forms a close extracellular association with the nasal epithelium of its host, and severe infections can result in lesions [11]. Infected hosts experience clinical signs of disease including nasal exudate, possibly leading to lethargic behavior and loss of appetite [5, 11].

M. agassizii is coccoid to pleomorphic in shape, lacks a cell wall, and has a three-layer membrane (Table 1, Fig. 1). These microbes range in size under 1 μm [10, 11] and grow in culture at an optimal temperature of 30 °C, with an extremely slow growth rate [10, 12]. Mortality of M. agassizii occurs at temperatures above 37 °C [12], and it retains viability after prolonged periods of cold temperatures [6, 10], indicating that body temperatures experienced by its ectothermic hosts likely affect the microbe’s success over the seasons. In an experiment to detect co-infection patterns of M. agassizii with its close relative M. testudineum , there was some indication that the two species form a facilitative relationship in a host-context-dependent manner [13]. Preliminary microbiome data suggest that the presence of M. agassizii is associated with a shift in the microbial community composition in Mojave and Sonoran Desert tortoises ( Gopherus morafkai ) (CLW, FCS and CRT, unpublished data).
Table 1

Classification and general features of Mycoplasma agassizii, strains PS6T and 723

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [38]

  

Phylum Firmicutes

TAS [39]

  

Class Mollicutes

TAS [40]

  

Order Mycoplasmatales

TAS [41, 42]

  

Family Mycoplasmataceae

TAS [42]

  

Genus Mycoplasma

TAS [10]

  

Species Mycoplasma agassizii

TAS [10]

  

Strains PS6T and 723

TAS [5, 6, 10, 20]

 

Gram stain

Negative

NAS

 

Cell shape

Coccoid to pleomorphic

TAS [10]

 

Motility

Non-motile

TAS [10]

 

Sporulation

Nonspore-forming

NAS

 

Temperature range

Not reported

 
 

Optimum temperature

30 °C

TAS [10]

 

pH range; Optimum

Not reported

 
 

Carbon source

Glucose

TAS [10]

MIGS-6

Habitat

Tortoise respiratory tract

TAS [10]

MIGS-6.3

Salinity

Not reported

 

MIGS-22

Oxygen requirement

Aerobic

TAS [10]

MIGS-15

Biotic relationship

Symbiont

TAS [11]

MIGS-14

Pathogenicity

Pathogenic

TAS [5, 6]

MIGS-4

Geographic location

North America

TAS [6, 10]

MIGS-5

Sample collection

1991 (PS6T), 1992 (723)

TAS [43]

MIGS-4.1

Latitude

Approx.: 36 N (PS6T), 26.4 N (723)

TAS [6, 10]

MIGS-4.2

Longitude

Approx.: 115 W (PS6T), 82.1 W (723)

TAS [6, 10]

MIGS-4.4

Altitude

Approx.: 800 m (PS6T), 0 m (723)

TAS [6, 10]

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 [44]

aEvidence codes

Figure 1
Fig. 1

Electron micrograph of ultrathin section of Mycoplasma agassizii strain PS6T. Image from ref. [10], reproduced with permission from the publisher. Scale bar = 0.5 μm

The strains of M. agassizii that we have sequenced were isolated from two host species. Strain PS6T was isolated from the upper respiratory tract of a Mojave Desert tortoise in the Las Vegas Valley, Nevada, USA [10], while strain 723 was obtained from an ill gopher tortoise in Sanibel Island, Florida, USA [6]. Strains were cultured in SP4 broth, and have been used in experiments to demonstrate their pathogenic effects on their tortoise hosts [5, 6].

To determine the placement of M. agassizii in the mycoplasmal phylogeny, all 16S rRNA gene sequences from the type strains of Mycoplasma species were obtained from the SILVA database [14] and aligned using MUSCLE 3.8.31 [15], and a phylogenetic tree was constructed using the maximum likelihood method implemented in MEGA7 [16] (Fig. 2). Consistent with prior results [17, 18], M. testudineum is a sister group of M. agassizii in the resultant tree, and the M. agassizii / M. testudineum clade is a sister group of Mycoplasma pulmonis , the agent of murine respiratory mycoplasmosis. All three species fall within the hominis group of Mycoplasma (see ref. [19] for group definitions). The 16S rRNA gene sequence from M. agassizii , strain PS6T, is 99.8, 93.2 and 89.2% identical to those of M. agassizii strain 723, M. testudineum strain BH29T, and M. pulmonis strain PG34T, respectively.
Figure 2
Fig. 2

Phylogenetic tree of the Mycoplasma genus based on 16S rRNA gene sequences showing the phylogenetic position of M. agassizii PS6T and 723 (). All 16S sequences from the Mycoplasma genus were obtained from the SILVA database [14]. Only sequences in the ‘The All-Species Living Tree’ Project (LTP), release 128, were retained. This dataset only contains sequences from type strains, designated with a superscripted “T”. Clostridium botulinum strain ATCC 25763 was also included in the dataset as outgroup. Sequences were aligned using MUSCLE 3.8.31 [15]. A phylogenetic tree was obtained using the maximum likelihood method implemented in MEGA7 [16], with 1000 bootstrap replicates. Species with available genomes at the NCBI Genomes database or the Genomes Online Database are represented in bold face. GenBank accession numbers are shown in parentheses. Bootstrap support values above 50% are represented. The scale bar represents a divergence of 0.05 nucleotide substitutions per nucleotide position

Genome sequencing information

Genome project history

Two strains of M. agassizii were selected for sequencing, strains PS6T and 723, both isolated from tortoises with signs of URTD [5, 6, 10, 20]. Sequencing was conducted in October 2016. The Whole Genome Shotgun projects were deposited at DDBJ/ENA/GenBank under the accession numbers NQMN00000000 (strain PS6T) and NQNY00000000 (strain 723). The versions described in this paper are the first versions. A summary of the information of both projects in compliance with MIGS version 2.0 [21] is shown in Table 2.
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High quality drafts

MIGS-28

Libraries used

Illumina Nextera XT

MIGS-29

Sequencing platforms

Illumina NextSeq500

MIGS-31.2

Fold coverage

38.51 (strain PS6T); 37.73 (strain 723)

MIGS-30

Assemblers

SPAdes 3.10.1

MIGS-32

Gene calling method

NCBI Prokaryotic Genome Annotation Pipeline 4.2

 

Locus Tag

CJF60 (strain PS6T); CJJ23 (strain 723)

 

GenBank ID

NQMN00000000 (strain PS6T); NQNY00000000 (strain 723)

 

GenBank Date of Release

August 28, 2017 (strain PS6T); August 29, 2017 (strain 723)

 

GOLD ID

 

BIOPROJECT

PRJNA397947 (strain PS6T); PRJNA398096 (strain 723)

MIGS-13

Source Material Identifier

ATCC 700616 (strain PS6T); ATCC 700617 (strain 723)

 

Project relevance

Animal parasite

Growth conditions and genomic DNA preparation

Freeze-dried M. agassizii strains were obtained from the ATCC in March 2011 (strain PS6T) and May 2016 (strain 723). Strain PS6T was cultured on SP4 media and re-pelleted in-lab prior to DNA extraction. Genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit protocol for Gram-negative bacteria and eluted with water. Extracted DNA was quantified on a Qiagen QIAxpert system and with Picogreen analysis.

Genome sequencing and assembly

Genome sequencing was conducted using the Illumina Nextera XT DNA Library Preparation Kit (Illumina, Inc., San Diego, USA) with the Illumina NextSeq500 platform (150 bp, paired-end) and 2 ng of starting genomic DNA at the Nevada Genomics Center (University of Nevada, Reno). Sequencing was performed in multiplex with multiple samples, using dual index sequences from the Illumina Nextera XT Index Kit, v2 (PS6 indices: index 1 N702, index 2 S510; 723 indices: index 1 N702, index 2 S511). A total of 349,251 and 332,967 read pairs were obtained for strains PS6T and 723, respectively. Using Trimmomatic, version 0.36 [22], reads were trimmed to remove Nextera adapter sequences and low quality nucleotides from either end (average Phred score Q ≤ 5, four bp sliding window), and sequences trimmed to < 35 bp were removed. After trimming, 330,351 (PS6T) and 305,002 (723) read pairs, and 16,438 (PS6T) and 25,017 (723) single-reads (the pairs of which were removed) remained. De novo genome assembly was performed using SPAdes 3.10.1 [23], using as inputs the trimmed paired reads, and the trimmed single reads (assembly k-mer sizes 21, 33, 55, and 77, with read error-correction enabled and ‘--careful’ mode mismatch correction). After removing scaffolds of less than 500 bp, the final assemblies consisted of 8 (PS6T) and 40 (723) scaffolds with a total length of 1,274,972 bp (PS6T) and 1,211,209 bp (723), an average length of 159,372 bp (PS6T) and 30,280 bp (723), and an N50 of 654,010 bp (PS6T) and 56,701 bp (723). The coverage was 38.51× for the PS6T assembly and 37.73× for the 723 assembly.

Genome annotation

Gene prediction was carried out using the NCBI Prokaryotic Genome Annotation Pipeline 4.2 [24]. For each predicted protein, (i) families were identified using the Pfam 31.0 [25] batch search tool (“gathering threshold” option), (ii) Clusters of Orthologous Groups categories were assigned using eggNOG-mapper [26] based on eggNOG 4.5.1 data [27], (iii) signal peptides were identified using the SignalP server 4.1 [28], and (iv) transmembrane helices were inferred using the TMHMM server v. 2.0 [29]. CRISPR repeats were identified using PGAP and CRISPRFinder [30].

Genome properties

The properties of both draft genomes are summarized in Table 3. The final assembly for strain PS6T consisted of 8 scaffolds, with a total length of 1,274,972 bp, and a G + C content of 28.43%. PGAP [24] identified a total of 979 protein-coding genes, 13 pseudogenes, and 35 RNA genes. The assembly for strain 723 consisted of 40 scaffolds, with a total length of 1,211,209 bp, and a G + C content of 28.34%. A total of 955 protein-coding genes, 7 pseudogenes, and 35 RNA genes were identified. In both cases, the identified RNA genes include 3 rRNAs (one 5S, one 16S and one 23S), 3 ncRNAs and 29 tRNAs. PGAP identified no CRISPR repeats in any of the two genomes, and CRISPRFinder [30] identified 6 “questionable” repeats in the PS6T genome and one “questionable” repeat in the 723 genome, but no “confirmed” repeats. The numbers of protein-coding genes in each COG category [31] were similar for both strains, and are summarized in Table 4.
Table 3

Genome statistics

Attribute

Strain PS6T

Strain 723

Value

% of Total

Value

% of Total

Genome size (bp)

1,274,972

100.00

1,211,209

100.00

DNA coding (bp)

1124,547a

88.20c

1072,218a

88.52c

DNA G + C (bp)

362,520

28.43c

343,241

28.34c

DNA scaffolds

8

100.00

40

100.00

Total genes

1027

100.00

997

100.00

Protein coding genes

979

95.33d

955

95.79d

RNA genes

35

3.41d

35

3.51d

Pseudo genes

13

1.27d

7

0.70d

Genes in internal clusters

Genes with function prediction

467b

47.70e

301b

31.52e

Genes assigned to COGs

581

59.35e

577

60.42e

Genes with Pfam domains

608

62.10e

607

63.56e

Genes with signal peptides

160

16.34e

150

15.71e

Genes with transmembrane helices

294

30.03e

288

30.16e

CRISPR repeats

0

0

aProtein-coding sequences, not including stop codons

bProteins not annotated as “hypothetical protein” by PGAP

cRelative to genome size

dRelative to total number of genes

eRelative to protein-coding genes

Table 4

Number of genes associated with general COG functional categories

Codea

Strain PS6T

Strain 723

Description

Valueb

%age

Valueb

%age

J

101

10.32

101

10.58

Translation, ribosomal structure and biogenesis

A

0

0.00

0

0.00

RNA processing and modification

K

21

2.15

20

2.09

Transcription

L

66

6.74

60

6.28

Replication, recombination and repair

B

0

0.00

0

0.00

Chromatin structure and dynamics

D

4

0.41

5

0.52

Cell cycle control, Cell division, chromosome partitioning

V

33

3.37

29

3.04

Defense mechanisms

T

5

0.51

5

0.52

Signal transduction mechanisms

M

10

1.02

10

1.05

Cell wall/membrane biogenesis

N

0

0.00

0

0.00

Cell motility

U

11

1.12

9

0.94

Intracellular trafficking and secretion

O

28

2.86

31

3.25

Posttranslational modification, protein turnover, chaperones

C

34

3.47

34

3.56

Energy production and conversion

G

72

7.35

74

7.75

Carbohydrate transport and metabolism

E

27

2.76

26

2.72

Amino acid transport and metabolism

F

25

2.55

25

2.62

Nucleotide transport and metabolism

H

13

1.33

13

1.36

Coenzyme transport and metabolism

I

9

0.92

9

0.94

Lipid transport and metabolism

P

35

3.58

36

3.77

Inorganic ion transport and metabolism

Q

1

0.10

1

0.10

Secondary metabolites biosynthesis, transport and catabolism

R

0

0.00

0

0.00

General function prediction only

S

92

9.40

93

9.74

Function unknown

398

40.65

378

39.58

Not in COGs

Percentages are based on the total number of protein coding genes in the genome

aCOG category code

bNumber of genes in the category

Insights from the genome sequence

The small genome size and low G + C content of both M. agassizii genomes described here are consistent with those of other Mycoplasma genomes sequenced [18, 32, 33]. However, the M. agassizii genomes are significantly larger than the genome of M. testudineum , strain BH29T (960,895 bp, 788 protein-coding genes; ref. [18]). The difference in the genome size of both sister species might account for the fact that M. agassizii is associated with URTD, whereas the link between M. testudineum and URTD is less clear [13]; i.e., genes present in M. agassizii but not in M. testudineum might be responsible for pathogenicity.

In spite of the fact that the two M. agassizii strains sequenced here were obtained from geographically distant locations (the Mojave Desert and Sanibel Island) and from different tortoise species ( G. agassizii and G. polyphemus ; refs. [5, 6, 10, 20]), the two genomes are very similar, exhibiting very similar sizes, numbers of genes (Table 3), functional composition (Table 4), and a high degree of synteny (Fig. 3a). A best-reciprocal-hit approach (based on BLASTP searches, E-value ≤10− 10) identified 828 pairs of putative orthologs within both genomes. The sequences of proteins encoded by pairs of orthologous genes were aligned using ProbCons version 1.12 [34], and were 93.57% identical on average (median: 96.84%). In contrast, comparison of the genomes of M. agassizii strain PS6T and M. testudineum strain BH29T [18] revealed much less synteny (Fig. 3b) and protein identity (average: 54.78%, median: 54.71%).
Figure 3
Fig. 3

Comparison of the genomes of M. agassizii strains PS6T and 723 (a), and M. agassizii strain PS6T and M. testudineum strain BH29T (b). The figure was generated using Circoletto 07.09.16 [45], a web interface for Circos [46]. The relative order of scaffolds is unknown. For strain PS6T, scaffolds are sorted by size

The 16S rRNA gene sequences of M. agassizii , strains PS6T and 723, differed at 3 nucleotide positions (Fig. 4). Surprisingly, our 16S sequence for strain PS6T and that obtained by Brown et al. (also for strain PS6T; ref. [20]) exhibit 8 differences (4 point differences and 4 indels; Fig. 4). These differences may represent mutations accumulated since the isolation of the strain, or sequencing errors.
Figure 4
Fig. 4

Comparison of the 16S rRNA gene sequences generated by Brown et al. [20] (M. agassizii strain PS6T; GenBank accession: U09786) and in our study (M. agassizii strains PS6T and 723). Asterisks represent identical sites

To initiate pathogenesis, Mycoplasma cells usually require adhering to the host mucosa. Adhesion mechanisms are relatively well understood in Mycoplasma pneumoniae and its close relatives, but poorly understood in other Mycoplasma species [35]. In a prior study, we searched all available Mycoplasma genomic data (nr database, including the genome of M. testudineum BH29T) for homologs of M. pneumoniae cytadhesins P1, P30, P65, P40 and P90 and cytadhesin accessory proteins hmw1, hmw2 and hmw3, finding homologs only in species closely related to M. pneumoniae ( Mycoplasma genitalium , Mycoplasma gallisepticum , Mycoplasma pirum , Mycoplasma alvi , Mycoplasma imitans , and Mycoplasma testudinis ) [18]. Here, we expanded these analyses (BLASTP and TBLASTN searches; E < 10− 5 and low-complexity regions filtering) to the two M. agassizii proteomes/genomes, also with negative results. In addition, none of the predicted M. agassizii proteins exhibit any of the Pfam domains present in the M. pneumoniae (domains “CytadhesinP1”, “Adhesin_P1”, “Cytadhesin_P30”, “MgpC” and “EAGR_box”). This could be attributed either to (i) M. pneumoniae adhesion proteins being specific to this species and its close relatives, or (ii) adhesion proteins evolving very fast, perhaps due to co-evolutionary races, precluding detection of homologs in distantly related species. The first possibility is supported by the fact that M. pulmonis , the most closely related known species to the M. agassizii / M. testudineum clade, exhibits adhesion mechanisms different from M. pneumoniae , lacking an attachment organelle [36]. In support of the second scenario, our analysis of orthologous sequences revealed poor protein conservation among the sister groups M. agassizii and M. testudineum .

We repeated our similarity searches using as query a list of known Mycoplasma adhesins, which we obtained by searching the text “ Mycoplasma adhesin” in the UniProt database [37]. Our prior searches against the M. testudineum BH29T proteome/genome failed to detect any significant hits. In the current study, we detected significant similarity between a Mycoplasma mobile protein annotated as a “Truncated adhesin protein” (UniProt ID: Q8L3E5_9MOLU) and the proteins CJF60_05070 (strain PS6T, 3308 amino acids) and CJJ23_03020 (strain 723, also 3308 amino acids) of M. agassizii . CJF60_05070 and CJJ23_03020 are 92% identical. The C-terminal part of the M. mobile protein exhibits homology to three regions of the M. agassizii proteins (positions 958–1261, 1296–1597 and 1717–1924 of CJF60_05070; positions 956–1259, 1294–1595 and 1715–1922 of CJJ23_03020). A BLASTP search using CJF60_05070 as query sequence against the nr database identified a total of 17 hits, including three adhesion proteins (Table 5). Of note, the first hit is a M. testudineum protein (34%), which was not detected in our prior analyses [18]. Equivalent results were obtained using the CJJ23_03020 protein sequence as query (data not shown). The TMHMM server v. 2.0 [29] predicted both CJF60_05070 and CJJ23_03020 to contain a transmembrane domain at the N-terminal part of the protein (positions 7–29), and most of the protein (positions 30–3308) to be extracellular. Taken together, these observations point to these proteins as potential M. agassizii adhesins.
Table 5

Results of a BLASTP search using CJF60_05070 as query against the nr database

Accession

Description

Total score

Query cover

E-value

Identity

WP_094254640.1

hypothetical protein [Mycoplasma testudineum]

1254

98%

0.0

34%

CAC13384.1

unknown; predicted coding region [Mycoplasma pulmonis]

683

98%

0.0

27%

WP_041363975.1

hypothetical protein [Mycoplasma pulmonis]

682

98%

0.0

26%

WP_011264623.1

Gli349 adhesion and gliding protein [Mycoplasma mobile]

310

67%

10−80

25%

CCY45197.1

fNIP repeat-containing protein [Clostridium sp. CAG:1193]

105

2%

2 × 10−4

38%

WP_015135277.1

hypothetical protein [Leptolyngbya sp. PCC 7376]

215

5%

3 × 10−4

34%

AET68682.1

conserved repeat protein [Desulfosporosinus orientis DSM 765]

58.5

3%

3 × 10−4

38%

OPH56032.1

hypothetical protein BC351_29535 [Paenibacillus ferrarius]

105

5%

4 × 10−4

35%

KRK80309.1

adhesion exoprotein [Lactobacillus nodensis DSM 19682 = JCM 14932 = NBRC 107160]

57.8

2%

5 × 10−4

40%

WP_081776155.1

hypothetical protein [Lactobacillus nodensis]

57.4

2%

6 × 10−4

40%

CCY44912.1

fNIP repeat-containing protein [Clostridium sp. CAG:1193]

55.1

3%

6 × 10−4

35%

WP_057878036.1

hypothetical protein [Lactobacillus paucivorans]

53.5

4%

0.010

30%

WP_066545473.1

hypothetical protein [Caryophanon tenue]

53.1

5%

0.012

29%

WP_081780332.1

hypothetical protein [Porphyromonas uenonis]

97.8

2%

0.150

37%

BAB92076.1

truncated adhesin protein [Mycoplasma mobile]

47.4

9%

0.770

24%

Conclusions

We have obtained draft genome sequences for M. agassizii , strains PS6T and 723, both isolated from tortoises of the genus Gopherus with URTD. Both genomes exhibited a very small size and low G + C content, which is typical from Mycoplasma genomes. The two assemblies were very similar, in terms of synteny and protein sequences, in spite of the fact that they were obtained from different hosts and geographical locations. We identified a putative cytadhesin in both genomes. The new genomes will facilitate future studies that will help understand the molecular bases of pathogenicity of this and other Mycoplasma species.

Abbreviations

ATCC: 

American Type Culture Collection

BLAST: 

Basic local alignment search tool

COG: 

Clusters of Orthologous Groups

MIGS: 

Minimum information on the genome sequence

MRM: 

Murine respiratory mycoplasmosis

NCBI: 

National Center for Biotechnology Information

PGAP: 

Prokaryotic Genome Annotation Pipeline

URTD: 

Upper respiratory tract disease

Declarations

Acknowledgements

The authors are very grateful to Kris Kruse from the Nevada Genomics Center for technical assistance, and to Marco Fondi for helpful discussions. They are also grateful to the Nevada Genomics Center for providing sequencing services for free.

Funding

This work was made possible by a grant from the National Institute of General Medical Sciences (P20GM103440) from the National Institutes of Health. The funder did not play any role in the study.

Authors’ contributions

DAP, CLW, FCS and CRT conceived the work. CLW conducted laboratory work. DAP and RLT conducted bioinformatic analyses. DAP and CLW drafted the manuscript. All authors contributed to interpreting data and improving the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Biology Department, University of Nevada, Reno, USA
(2)
Nevada Center for Bioinformatics, University of Nevada, Reno, USA
(3)
Biology Department, Colorado State University, Pueblo, USA

References

  1. Brown DR. Mycoplasmosis and immunity of fish and reptiles. Front Biosci. 2002;7:d1338–46.View ArticlePubMedGoogle Scholar
  2. Seigel RA, Smith RB, Seigel NA. Swine flu or 1918 pandemic? Upper respiratory tract disease and the sudden mortality of gopher tortoises (Gopherus polyphemus) on a protected habitat in Florida. J Herpetol. 2003;37(1):137–44.View ArticleGoogle Scholar
  3. McCoy ED, Mushinsky HR, Lindzey J. Conservation strategies and emergent diseases: the case of upper respiratory tract disease in the gopher tortoise. Chelonian Conserv Biol. 2007;6(2):170–6.View ArticleGoogle Scholar
  4. Desert Tortoise Recovery Team. Desert tortoise (Mojave population): recovery plan. Portland: US Fish and Wildlife Service; 1994.Google Scholar
  5. Brown MB, Schumacher IM, Klein PA, Harris K, Correll T, Jacobson ER. Mycoplasma agassizii causes upper respiratory tract disease in the desert tortoise. Infect Immun. 1994;62(10):4580–6.PubMedPubMed CentralGoogle Scholar
  6. Brown M, McLaughlin G, Klein P, Crenshaw B, Schumacher I, Brown D, Jacobson E. Upper respiratory tract disease in the gopher tortoise is caused by mycoplasma agassizii. J Clin Microbiol. 1999;37(7):2262–9.PubMedPubMed CentralGoogle Scholar
  7. Brown D, Merritt J, Jacobson E, Klein P, Tully J, Brown M. Mycoplasma testudineum sp. nov., from a desert tortoise (Gopherus agassizii) with upper respiratory tract disease. Int J Syst Evol Microbiol. 2004;54(5):1527–9.View ArticlePubMedGoogle Scholar
  8. Sandmeier FC, Tracy CR, Hunter K. Upper respiratory tract disease (URTD) as a threat to desert tortoise populations: a reevaluation. Biol Conserv. 2009;142(7):1255–68.View ArticleGoogle Scholar
  9. Diemer Berish JE, Wendland LD, Kiltie RA, Garrison EP, Gates CA. Effects of mycoplasmal upper respiratory tract disease on morbidity and mortality of gopher tortoises in northern and Central Florida. J Wildl Dis. 2010;46(3):695–705.View ArticleGoogle Scholar
  10. Brown M, Brown D, Klein P, McLaughlin G, Schumacher IM, Jacobson E, Adams H, Tully J. Mycoplasma agassizii sp. nov., isolated from the upper respiratory tract of the desert tortoise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphemus). Int J Syst Evol Microbiol. 2001;51(2):413–8.View ArticlePubMedGoogle Scholar
  11. Jacobson ER, Gaskin J, Brown M, Harris R, Gardiner C, LaPointe J, Adams H, Reggiardo C. Chronic upper respiratory tract disease of free-ranging desert tortoises (Xerobates agassizii). J Wildl Dis. 1991;27(2):296–316.View ArticlePubMedGoogle Scholar
  12. Mohammadpour HA. Mycoplasma agassizii infections in the desert tortoise (Gopherus agassizii). PhD dissertation. Reno: University of Nevada; 2011.Google Scholar
  13. Weitzman CL, Gov R, Sandmeier FC, Snyder SJ, Tracy CR. Co-infection does not predict disease in Gopherus tortoises. Royal Soc Open Sci. 2017;4:171003.View ArticleGoogle Scholar
  14. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41(Database issue):D590–6.PubMedGoogle Scholar
  15. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–7.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4.View ArticlePubMedGoogle Scholar
  17. Volokhov DV, Simonyan V, Davidson MK, Chizhikov VE. RNA polymerase beta subunit (rpoB) gene and the 16S–23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae. Mol Phylogenet Evol. 2012;62(1):515–28.View ArticlePubMedGoogle Scholar
  18. Weitzman CL, Tillett RL, Sandmeier FC, Tracy CR, Alvarez-Ponce D. High quality draft genome sequence of Mycoplasma testudineum strain BH29T, isolated from the respiratory tract of a desert tortoise. Stand Genomic Sci. 2018;13:9.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Weisburg W, Tully J, Rose D, Petzel J, Oyaizu H, Yang D, Mandelco L, Sechrest J, Lawrence T, Van Etten J. A phylogenetic analysis of the mycoplasmas: basis for their classification. J Bacteriol. 1989;171(12):6455–67.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Brown D, Crenshaw B, McLaughlin G, Schumacher I, McKenna C, Klein P, Jacobson E, Brown M. Taxonomic analysis of the tortoise mycoplasmas mycoplasma agassizii and mycoplasma testudinis by 16S rRNA gene sequence comparison. Int J Syst Evol Microbiol. 1995;45(2):348–50.Google Scholar
  21. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26(5):541.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comp Biol. 2012;19(5):455–77.View ArticleGoogle Scholar
  24. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016;44(14):6614–24.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016;44(D1):D279–85.View ArticlePubMedGoogle Scholar
  26. Huerta-Cepas J, Forslund K, Pedro Coelho L, Szklarczyk D, Juhl Jensen L, von Mering C, Bork P. Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol Biol Evol. 2017;34(8):2115–22.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Huerta-Cepas J, Szklarczyk D, Forslund K, Cook H, Heller D, Walter MC, Rattei T, Mende DR, Sunagawa S, Kuhn M. eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 2015;44(D1):D286–93.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8(10):785–6.View ArticlePubMedGoogle Scholar
  29. TMHMM Server v. 2.0. http://www.cbs.dtu.dk/services/TMHMM/. Accessed Aug 2017.
  30. Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35(suppl_2):W52–7.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, Krylov DM, Mazumder R, Mekhedov SL, Nikolskaya AN, et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003;4:41.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Fraser CM, Gocayne JD, White O, Adams MD, Clayton RA, Fleischmann RD, Bult CJ, Kerlavage AR, Sutton G, Kelley JM, et al. The minimal gene complement of mycoplasma genitalium. Science. 1995;270(5235):397–403.View ArticlePubMedGoogle Scholar
  33. Citti C, Blanchard A. Mycoplasmas and their host: emerging and re-emerging minimal pathogens. Trends Microbiol. 2013;21(4):196–203.View ArticlePubMedGoogle Scholar
  34. Do CB, Mahabhashyam MS, Brudno M, Batzoglou S. ProbCons: probabilistic consistency-based multiple sequence alignment. Genome Res. 2005;15(2):330–40.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Browning GF, Noormohammadi AH, Markham PF. Identification and characterization of virulence genes in mycoplasmas. In: Browning GF, Citti C, editors. Mollicutes: molecular biology and pathogenesis. Norfolk: Caister Academic Press; 2014. p. 77–90.Google Scholar
  36. Cassell GH. The pathogenic potential of mycoplasmas: mycoplasma pulmonis as a model. Rev Infect Dis. 1982;4(Supplement_1):S18–34.View ArticlePubMedGoogle Scholar
  37. Uniprot Consortium. UniProt: a hub for protein information. Nucleic Acids Res. 2015;43(Database issue):D204–12.View ArticleGoogle Scholar
  38. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87(12):4576–9.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer K-H, Whitman W. Bergey’s manual of systematic bacteriology: volume 3: the Firmicutes. 2nd ed. New York: Springer-Verlag; 2011.Google Scholar
  40. Whitcomb R, Tully J, Bové J, Bradbury J, Christiansen G, Kahane I, Kirkpatrich B, Laigret F, Leach R, Neimank H. Revised minimum standards for description of new species of the class Mollicutes (division Tenericutes). J Syst Bacteriol. 1995;45:605–12.View ArticleGoogle Scholar
  41. Edward DGF, Freundt E. Type strains of species of the order Mycoplasmatales, including designation of neotypes for mycoplasma mycoides subsp. mycoides, mycoplasma agalactiae subsp. agalactiae, and mycoplasma arthritidis. Int J Syst Evol Microbiol. 1973;23(1):55–61.Google Scholar
  42. Freundt E. The classification of the pleuropneumonia group of organisms (Borrelomycetales). Int J Syst Evol Microbiol. 1955;5(2):67–78.Google Scholar
  43. American Type Culture Collection Catalog. https://www.atcc.org. Accessed Oct 2017.
  44. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet. 2000;25(1):25–9.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Darzentas N. Circoletto: visualizing sequence similarity with Circos. Bioinformatics. 2010;26(20):2620–1.View ArticlePubMedGoogle Scholar
  46. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19(9):1639–45.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© The Author(s). 2018

Advertisement