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Biotechnology Letters

, Volume 40, Issue 2, pp 335–341 | Cite as

Species of family Promicromonosporaceae and family Cellulomonadeceae that produce cellulosome-like multiprotein complexes

  • Wei Wang
  • Yang Yu
  • Tong-Yi Dou
  • Jia-Yue Wang
  • Chenggong Sun
Original Research Paper

Abstract

Objectives

To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights.

Results

Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit.

Conclusion

This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular β-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae.

Keywords

Cellulomonadeceae Cellulosome Multienzyme complexes Promicromonosporaceae β-Xylosidase 7-Xylosyltaxanes 

Introduction

The genus Cellulosimicrobium, together with its closest phylogenetic neighbors Isoptericola, Xylanimonas, Xylanimicrobium, and Xylanibacterium, is reclassified as a member of family Promicromonosporaceae, belonging to the order Micrococcales (Stackebrandt and Prauser 1991, Whitman et al. 2012).

The type species, Cellulosimicrobium cellulans, formerly known as Cellulomonas cellulans, includes the non-motile strains of Nocardia cellulans, Brevibacterium lyticum, and the motile strains of Arthrobacter luteus, Oerskovia xanthineolytica for their phylogenetic and chemotaxonomic relatedness (Schumann et al. 2001). This species was first isolated and characterized from chalk grassland soil by Metcalfe and Brown (1957), and was noted for its ability to fix N2 with cellulose as carbon source. Several commercially-available yeast-lytic glucanase preparations derived from this organism, namely Lyticase, Zymolyase, and Quantazyme, are still widely used for yeast protoplast preparation and yeast DNA isolation (Kitamura et al. 1971; Palumbo et al. 2003; Ferrer 2006).

When screening for glycoside hydrolases that can specifically remove the C-7 xylosyl group from 7-xylosyl-10-deacetylpaclitaxel (DAXP), a valuable semisynthetic precursor of Taxol (Jordan and Wilson 2004), a Cellulosimicrobium cellulans wild type strain F16 had the highest activity (Hao et al. 2008, Dou et al. 2015a). Further studies revealed that the extracellular β-xylosidase attributed to a cellulosome-like, non-cellulolytic multienzyme complex with an ellipsoid morphology under transmission electron microscopy (Dou et al. 2015b). As previously documented, cellulosomes are produced by anaerobic microorganisms (Bayer et al. 2008). Therefore, our findings seem to be unusual and surprising. Accordingly, we have applied zymography analysis to investigate whether all the phylogenetically related species and genera of Cellulosimicrobium cellulans, covering both the family Promicromonosporaceae (to which Cellulosimicrobium cellulans currently belongs) and the family Cellulomonadeceae (to which it previously belonged), also produce such a nanoscale protein complex.

Here we report that the genus Cellulosimicrobium and nearly all the genera of the family Cellulomonadeceae can produce both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes, while the other genera of the family Promicromonosporaceae cannot. Fermentation and zymography were performed to determine the above differences. Homology alignments of the key subunit were performed to get molecular insights into the above phenomenon.

Materials and methods

Strains used in this work

22 strains were studied. Most were typical strains of the phylogenetically-related species and genus of Cellulosimicrobium cellulans, covering both the family Promicromonosporaceae and the family Cellulomonadeceae, order Micrococcales (Whitman et al. 2012). Sources of these strains are listed in Table 1.
Table 1

Sources of the strains studied in this work

 

Nomenclature

16S rRNA accession no.

CGMCC

JCM

ATCC

1

Cellulosimicrobium cellulans

X79455

JCM 9965

ATCC 12830

2

Cellulosimicrobium cellulans

EU287931

Strain F16 from this lab (reference)

3

Cellulosimicrobium terreum

 

JCM 15619

4

Cellulosimicrobium funkei

 

JCM 14302

ATCC BAA-886

5

Isoptericola hypogeus

AJ854061

JCM 15589

6

Isoptericola variabilis

AJ298873

JCM 11754

ATCC BAA-303

7

Isoptericola dokdonensis

DQ387860

JCM 15137

8

Isoptericola halotolerans

AY789835

JCM 13590

9

Xylanimonas cellulosilytica

AF403541

JCM 12276

10

Xylanibacterium ulmi

AY273185

JCM 14284

11

Xylanibacterium pachnodase

AF105422

JCM 13526

12

Myceligenerans xiligouense

AY354285

1.3458

JCM 14112

13

Promicromonospora citrea

 

4.1485

JCM 3051

ATCC 15908

14

Promicromonospora sukumoe

AB056130

JCM 6845

15

Promicromonospora aerolata

AJ487303

JCM 14119

16

Promicromonospora vindobonensis

AJ487302

JCM 14120

17

Cellulomonas fimi

X83803

1.19

JCM 1341

ATCC 484

18

Cellulomonas flavigena

X83799

1.1914

JCM 18109

ATCC 482

19

Oerskovia enterphila

X83807

JCM 7350

20

Oerskovia turbata

X79454, X83806

4.119

JCM 3160

ATCC 25835

21

Demequina aestuarii

DQ010160

JCM 12123

22

Actinotalea fermentans

X79458, X83805

JCM 9966

ATCC 43279

Cell culture and enzyme detection

Cell culture, enzyme detection and zymography analysis were performed as reported by Dou et al. (2015a). Ball mill-crushed corn cob was used as sole carbon source in the culture medium. Cells were grown in a 250 ml flask containing 50 ml culture medium with shaking at 150 rpm and at 30 °C for 96 h. Sampling was conducted daily, and test samples were stored at − 80 °C. Enzyme activity assays were performed by detecting the deglycosylated product, 10-deacetylpaclitaxel (DAP), using HPLC. Zymography analysis were performed using 2–15% continuous gradient non-denaturing polyacrylamide gels without spacer. After electrophoresis, gels were firstly incubated with 0.5 mM methyllumbelliferyl β-D-xyloside (MUX, in 50 mM pH 7.5 Tris/HCl buffer) at 30 °C for 5 min, and then viewed and photographed under ultraviolet light (365 nm). Finally, gels were restained again with Coomassie Brilliant Blue R-250.

Results and discussion

Strains that produce extracellular DAXP β-xylosidase activity and cellulosome-like multiprotein complex.

Among the 22 strains examined, only nine, including Cellulosimicrobium cellulans strain F16 (Dou et al. 2015a), produced an extracellular β-xylosidase active against DAXP (Fig. 1). Electrophoresis and zymography analysis of the cell-free culture supernatant revealed that eight strains, Actinotalea fermentans JCM 9966T, Cellulosimicrobium funkei JCM 14302T, Oerskovia turbata JCM 3160T, Cellulosimicrobium terreum JCM 15619T, Demequina aestuarii JCM 12123T, Cellulomonas flavigena JCM 18109T, Cellulosimicrobium cellulans JCM 9965T, and Oerskovia enterphila JCM 7350T, produce cellulosome-like complexes with β-xylosidase activity (Fig. 2). This is similar to the previous report for strain F16 by Dou et al. (2015b). Isoptericola dokdonensis JCM 15137 only produced “smaller” β-xyloside hydrolytic protein bands, compared to the cellulosome-like complex observed, yet no DAXP β-xylosidase activity was detected.
Fig. 1

Extracellular β-xylosidase activity against 7-xylosyl-10-deacetylpaclitaxel (DAXP). One unit of the enzyme activity was defined as the amount of enzyme that can release 1 μmol of the deglycosylated product DAP per minute, in 50 mM Tris/HCl buffer, pH 7.5, at 30 °C. Columns filled in black, light grey, dark grey, and white indicate enzyme activity of samples collected at 24 h, 36 h, 72 h, and 96 h, respectively. Mean values and standard deviations of triplicates are presented

Fig. 2

Electrophoresis and zymogram results a, c, e: Coomassie blue staining; b, d, f: in gel staining using 4-methyllumbelliferyl β-D-xyloside (MUX) as the fluorescence probe. For af, lane 1 referred to a mixture marker of BSA (66.5 kDa), BSA dimer (133.0 kDa), and ferritin (440 kDa). Lane 2–9 of a and b indicate cell-free culture produced by strain JCM 9965, JCM 15589, JCM 11754, JCM 15137, JCM 13590, JCM 12276, JCM 14284, JCM 13526; lane 2–9 of c and d indicate cell-free culture produced by strain JCM 9965, JCM 14112, JCM 3051, JCM 6845, JCM 14119, JCM 14120, JCM 15619, JCM 14302; while lane 2–9 of e and f indicate cell-free culture produced by strain JCM 9965, JCM 1341, JCM 18109, JCM 7350, JCM 3160, JCM 12123, JCM 9966, and F16

None of the Promicromonosporaceae, except for Cellulosimicrobium, produced an extracellular DAXP β-xylosidase activity or the cellulosome-like complex. In contrast, almost all the genera of family Cellulomonadeceae produced both extracellular DAXP β-xylosidase and cellulosome-like complexes. This suggests convergent evolution of the metabolic phenotype. Degradations of xylan or cellulose are similar in both Cellulosimicrobium and members of the family Cellulomonadeceae, since they all produce cellulosome-like complexes. For other members of the family Promicromonosporaceae, however, no such complexes were detected under the same conditions.

Our results provide insights into the polysaccharide degradation systems of these species (Ferrer 2006). Cellulosimicrobium and the family Cellulomonadeceae have a plant cell-wall degradation system akin to the multienzyme system, the cellulosome (Doi and Kosugi 2004, Bayer et al. 2008). The family Promicromonosporaceae, except Cellulosimicrobium, may have a distinct strategy for polysaccharide degradation. Consequently, from the perspective of producing cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against DAXP, the genus Cellulosimicrobium is still closer to members of the Cellulomonadeceae family than to the Promicromonosporaceae family.

Doi and Kosugi (2004) and Bayer et al. (2008) reported that cellulosomes were only produced by anaerobic bacteria (a few species of the phylum Firmicutes and Bacteroidetes) and some anaerobic fungi. Aerobic Actinobacterium uses systems comprised of independently-acting cellulose-degrading enzymes, often with carbohydrate-binding domains (Koeck et al. 2014, Zhao et al. 2017). Our research is the first to report that the aerobic actinobacteria can produce cellulosome-like, nanoscale multienzyme complexes and may open a new venue for cellulosome or cellulosome-related research.

In addition, the detection of the cellulosome-like complexes by zymography (using MUX as the fluorescence probe) and the production of an extracellular β-xylosidase active against 7-xylosyltaxanes (see Fig. 3) can also be regarded as key features that differentiate the genus Cellulosimicrobium from other genera of family Promicromonosporaceae.
Fig. 3

Phylogenetic analyses of the two families. The GenBank accession no. of 16S rRNA gene sequences of the strains are listed in Table 1 (Benson et al. 2005). Sequences were aligned and a distance matrix was created with CLUSTAL W (Larkin et al. 2007). A phylogenetic tree was constructed in TREECON with the neighbor-joining method and bootstrapped with 1000 replications (McNeil et al. 2004). * Strains that produce cellulosome-like multienzyme complex and the extracellular β-xylosidase activity against DAXP

Molecular insights

Among the 22 strains studied, only 11 have their whole genome sequence data given in GenBank. This includes the whole genome shotgun (WGS) sequencing data of Cellulosimicrobium cellulans strain F16 reported by us (Dou et al. 2015b). The key subunit of the extracellular DAXP β-xylosidase produced by strain F16, namely M768_06655, was applied to search against the remaining nine genome data (Table 2 and also Supplementary Fig. 1). Five of the ten genome sequences harbored predicted proteins that were closely identical to M768_06655,. while JCM 3164, JCM 14302, and JCM 3160 produced an extracellular β-xylosidase active against DAXP (Fig. 1, strain J36 was not tested). Unsurprisingly, these three strains also produce cellulosome-like complexes.
Table 2

Genomic analysis of the related strains The amino acid sequence of the key catalytic subunit M768_06655 (GenBank accession number: KON73778.1) was used to search against the released genome data of the actinobacteria studied in this work by the NCBI Blast tool (Boratyn et al. 2013)

Strain

Genome accession

Key subunit identified (accession no.)

Identity (%)

Active to DAXP?

Cellulosome-like complex production?

Cellulosimicrobium cellulans F16

GCA_001274015.1 (this project)

KON73778.1

100%

Yes

Yes

Cellulosimicrobium cellulans J36

GCA_000526595.1

WP_024839223.1

89%

Unclear

Unclear

Cellulosimicrobium cellulans LMG16121

GCA_000321105.1

WP_081600039.1

81%

Yes

Yes

Cellulosimicrobium funkei NBRC 104118

GCA_001570825.1

WP_082774695.1

83%

Yes

Yes

Isoptericola variabilis 225

GCA_000215105.1

WP_013839314.1

69%

N.D.

N.D.

Xylanimonas cellulosilytica DSM 15894

GCA_000024965.1

N.D.

N.D.

N.D.

N.D.

Promicromonospora sukumoe DSM44121

GCA_000385135.1

WP_020017538.1

71%

N.D.

N.D.

Oerskovia turbata ASM71832v1

GCA_000718325.1

WP_030149893.1

70%

Yes

Yes

Actinotalea fermentans ATCC43279

GCA_001313965.1

N.D.

N.D.

Yes

Yes

Demequina aestuarii ASM97509v1

GCA_000975095.1

N.D.

N.D.

Yes

Yes

Cellulomonas fimi ATCC484

GCA_000212695.1

N.D.

N.D.

N.D.

N.D.

Accession numbers of the matched proteins and the corresponding identities were recorded

N.D. not detected

We propose that all strains capable of producing extracellular β-xylosidases active against 7-xylosyltaxanes should have M768_06655 or its homolog (Dou et al. 2015b). However, the results shown here only partly support this hypothesis. The reason may be incomplete genomic data of these strains or incorrect annotation of the genome data. Therefore, M768_06655 is probably the essential subunit for the assembly of the cellulosome-like multienzyme complexes, but future work is warrant to ascertain.

Notes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 31600641) and the Fundamental Research Funds for the Central Universities (No. DUT16RC(3)016).

Supporting Information

Supplementary Fig. 1—Multiple sequence alignment results (related to Table 2).

Supplementary material

10529_2017_2469_MOESM1_ESM.docx (23 kb)
Supplementary material 1 (DOCX 22 kb)

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© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.The Second Affiliated Hospital of Dalian Medical UniversityDalianChina
  2. 2.School of Life Science and MedicineDalian University of TechnologyPanjinChina
  3. 3.Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalianChina

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