Fig. S2. Unrooted neighbor-joining protein tree of proteolytic enzymes obtained from
karst water biofilm and tufa (A). The tree includes representative members of serine
protease families S1B, S1C, and S8A retrieved from MEROPS database (Rawlings
et al., 2012). Amino acid sequences of published proteases were retrieved from
GenBank and accession numbers are given in brackets. The length of branches
indicates the number of amino acid substitutions per site. Evolutionary analyses were
conducted using MEGA5 (Tamura et al., 2011). Bootstrap values were calculated
from 1,000 resamplings. Characteristics of protease-encoding genes pwb1 to pwb5
and the corresponding gene products (B).
Construction
of
fosmid
libraries
and
screening
for
proteolytic
enzymes
Bacterial strains and vectors used in the present study are shown in Supplementary
Table S1. Escherichia coli strains were routinely grown in Luria-Bertani (LB) medium
at 37°C. Escherichia coli strain EPI300-T1R (Epicentre Biotechnologies, Madison, WI,
USA) was used as a host for the cloning of metagenomic DNA. In addition, E. coli
strain TOP10 (Invitrogen) was employed for subcloning of the target genes. The
large-insert metagenomic fosmid libraries WB3, WB4, WB5 and WB5tufa (see
Supplementary Table S2) were constructed by using the CopyControl Fosmid Library
Production kit (Epicentre Biotechnologies) as described by Nacke et al. (Nacke et al.
2011b) resulting in approximately 36,800 library-containing clones (approximately
9,200 per sample), which were arrayed and stored in 96-well microtiter plates.
For activity-based screening, the arrayed library-containing E. coli clones were
streaked on LB agar plates containing skim milk (2% [wt/vol]) as indicator substrate.
To maintain the presence of recombinant fosmids and increase the copy number of
the fosmids the indicator agar contained 12.5 mg chloramphenicol L-1 and 0.001%
arabinose, respectively. Proteolytic activity exhibiting clones were identified by the
formation of halos on indicator agar after incubation for 1 to 14 days. Initially, 37
proteolytic clones were detected and controlled for unique fosmid inserts by
restriction
analysis
with
BamHI
(Fermentas,
St.
Leon-Rot,
Germany)
as
recommended by the manufacturer.
Sequence analysis of proteolytic activity conferring genes
To avoid time-consuming sequencing of complete fosmid inserts and to enable rapid
detection of proteolytic activity conferring genes the target genes were subcloned.
The recombinant fosmids from positive clones were isolated, sheared by sonication
for 3 s at 30% amplitude, cycle 0.5 (UP200S Sonicator, Dr. Hielscher GmbH).
Subsequently, the resulting DNA fragments were separated by agarose gel
electrophoresis. Appropriate fragments (1.5 to 3.5 kbp) were excised and purified
from gels by using the peqGold gel extraction kit (Peqlab Biotechnologie GmbH). The
resulting DNA fragments were ligated into pCR-2.1-TOPO (Invitrogen), and used to
transform E. coli TOP10 (Invitrogen) as recommended by the manufacturer. The
resulting recombinant E. coli strains were re-screened on the indicator agar. The
recombinant plasmids derived from positive clones were sequenced. The initial
prediction of ORFs located on the plasmids-inserts was performed by using the ORFfinder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) provided by the National
Center for Biotechnology Information (NCBI) and finally annotated with Artemis
(Rutherford et al. 2000). The results were verified and improved manually by using
criteria such as the presence of a ribosome-binding site, GC frameplot analysis, and
similarity to known genes. Classification of the identified proteases was performed by
BLAST
searches
implemented
in
the
MEROPS
peptidase
database
(http://merops.sanger.ac.uk) (Rawlings et al. 2012). Similarity searches of related
protein sequences were performed by employing BLAST against the GenBank
database. Domain structures were analyzed by submitting protein sequences to
InterProScan (Quevillon et al. 2005). Multiple alignments of deduced protein
sequences and construction of a phylogenetic tree were performed by employing
MEGA5 (Tamura et al. 2011).
Analyses of novel proteases
The functional analysis revealed that protein metabolism including proteindegradation is of importance in the biofilm community. The screening of
approximately 36,800 clones yielded 37 positive clones conferring a stable proteolytic
phenotype. Finally, five unique proteolytic activity-conferring genes (pwb1 to pwb5)
were identified. The deduced protein sequences revealed 50 to 87% amino acid
identity to amino acid sequences from known proteases (Figure S2B). Classification
of the deduced amino acid sequences according to the MEROPS protease database
(Rawlings et al. 2012) revealed that the enzymes belong to S1 (chymotrypsin family)
and S8 (subtilisin family) of the serine proteases. Proteases of family S1 are
endoproteases (Rawlings et al. 2012), which are often characterized by the presence
of an N-terminal signal peptide and a propeptide. Interestingly, Pwb1 belongs to
subfamily S1B and the corresponding gene encodes no signal peptide, which has
been also observed for a protease (CBM43238) detected in a study focused on
screening of proteases from different environmental samples of Germany (Niehaus et
al. 2011). Pwb2 belongs to subfamily S1C and is a putative periplasmic enzyme with
a signal peptide showing highest identity to a hypothetical protein of eukaryotic origin.
Pwb3 and Pwb5 grouped into family subfamily S8A, which is represented by the
endoprotease subtilisin Carlsberg.
All deduced amino acid sequences, except Pwb2 exhibited highest similarities
to cyanobacterial proteases (Figure S2A). Pwb2 showed highest identity (50%) to a
hypothetical protein of the fern Selaginella moellendorffii. Due to the low similarity of
Pwb2 to the related database entry it is likely that this gene originated from
cyanobacteria or chloroplasts. The cyanobacterial-related proteases (Pwb1, Pwb3,
Pwb4 and Pwb5) were affiliated to filamentous Oscillatoriales (Leptolynbya sp. PCC
7375 and Oscillatoria nigro-viridis) and are probably derived from members of the
high abundant TPM clade detected by 16S rRNA gene analysis of the biofilm (Figure
5A).
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