CODIFICADORES/DECODIFICADORES (CODECS)

Bioinformatics tools were used to identify antimicrobial biosynthetic clusters and to predict the antimicrobial products encoded by these clusters (Table 2.1). These bioinformatic tools were also used for analysis of the genes disrupted by transposon mutagenesis (Sections 2.1.18 and 2.1.19). Similarities to other known genes and proteins were established by an alignment search tool using the NCBI BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990). Comparative genome analysis was represented using Easyfig (Sullivan et al., 2011).

Table 2.1. The bioinformatics analysis tools used to analyse the antimicrobial biosynthesis gene

clusters in P. c. subsp. brasiliensis ICMP 19477.

Name Description URL/Reference

Non-ribosomal peptide synthetase specific tools

PKS/NRPS Analysis Web- server

Blast server to identify homologues within the database plus domain organisation

http://nrps.igs.umaryland.edu/nrps/ (Bachmann and Ravel, 2009)

NRPS-PKS Prediction of domain

organisation and substrate specificity

URL no longer available (Ansari et al., 2004)

Antibiotics and secondary metabolites analysis shell (antiSMASH)

Identifies, annotates and analyses secondary metabolite producing loci. Also predicts the structure of the resulting product.

http://antismash.secondarymetabolites.org/ (Medema et al., 2011)

NRPSpredictor2 Predicts A domain specificity

and the resulting amino acid substrate

http://nrps.informatik.unituebingen.de/ (Rottig et al., 2011)

2.1.2

Chemicals and Media

All chemicals used in this study were of an analytical grade. The recipes for chemical solutions and media are given in Appendix A. Solutions were routinely prepared using double distilled water (ddH2O)

unless otherwise indicated. All chemical solutions and media were sterilized by autoclaving at 121°C (20 psi) for 20 min. Where appropriate, the pH of the solutions and media were measured at room temperature.

2.1.3

Bacterial strains and plasmids

Bacterial strains and plasmids are listed in Table 2.2.

Name Description URL/Reference

General tools used for analysis of antibiotic synthesis clusters

Pfam 26.0 Identification of protein family

similarities within the protein family sequence database

http://pfam.xfam.org/ (Finn et al., 2010)

HMMER Identification of protein

homologs and protein sequence alignments

http://hmmer.janelia.org (Finn et al., 2011)

BLAST-conserved domains Compares sequence with

databases to infer functional relationships.

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi (Altschul et al., 1990)

InterProScan 5 Blasts sequence against the

InterPro database of protein signatures to identify the domain structure.

http://www.ebi.ac.uk/Tools/pfa/iprscan (Jones et al., 2014)

I-TASSER server (Version 4.4)

Predicts the structure and function of protein sequences

http://zhanglab.ccmb.med.umich.edu/I- TASSER/

(Zhang, 2008) PredictProtein

(Version 1.0.88)

Secondary structure and structural annotations of protein sequences.

https://www.predictprotein.org/ (Rost et al., 2004)

Raptor X Protein secondary and tertiary

structure prediction

http://raptorx.uchicago.edu/ (Källberg et al., 2012)

Table 2.2. Bacterial strains and plasmids used in this study.

Antibiotic resistances are represented as follows: Km, Kanamycin; Rif, Rifampicin; Str, streptomycin; Amp, ampicillin; Chl, chloramphenicol; Tet, tetracycline

.

Bacterial Strain Description/Genotype Source/Reference Antibiotic

Resistance P. atrosepticum

SCRI1043 Wild Type JHI, UK N/A

SCRI1043 R Spontaneous genetic mutation conferring resistance to

rifampicin

This study Rif

SCRI1043 K SCRI1043 ECA0522Ar1::mTn5-gusA-pgfp::KmR _{(Holeva et al., 2004; Vanga et al.,}

2012)

Km

P. carotovorum subsp. carotovorum

ICMP 5702 Wild Type (Panda et al., 2015a) N/A

P. carotovorum subsp. brasiliensis

ICMP 19477 Wild Type (Pitman et al., 2008) N/A

ICMP 19477 R _{Spontaneous genetic mutation conferring resistance to}

rifampicin

This study Rif

ICMP 19477 K mTn5-gusA-gfp::KmR _{This study Km}

ICMP 19477 carR- _{carR::mTn5-gusA-gfp::Km}R _{This study Km}

ICMP 19477 carR-_{::pTRB32oriTcarR ICMP19477 carR}-_{containing pTRB32oriT containing a 738}

bp fragment, encompassing KCO_04647.

This study Chl, Km

ICMP 19477 carR-_{::pTRB32oriT ICMP19477 carR}-_{containing an empty copy of pTRB32oriT This study Chl, Km}

ICMP 19477 carI- _{carI::mTn5-gusA-gfp::Km}R _{This study Km}

ICMP 19477 slyA- _{slyA::mTn5-gusA-gfp::Km}R _{This study Km}

ICMP 19477 slyA-_{::pTRB32oriTslyA ICMP19477 slyA}-_{pTRB32oriT containing a 436 bp}

fragment, encompassing of KCO_21137.

This study Chl, Km

ICMP 19477 slyA-_{::pTRB32oriT ICMP19477 slyA}-_{containing an empty copy of pTRB32oriT This study Chl, Km}

ICMP 19477 abc- _{ICMP19477 carrying a single crossover mutation in ABC,}

integrated with pK18mobsacB.

(Preetinanda Panda, 2014; Doctoral thesis)

Chl, Km

ICMP 19477 nrps1- _{ICMP19477 carrying a double crossover mutation in nrps1}

integrated with pK18mobsacB.

(Preetinanda Panda, 2014; Doctoral thesis)

ICMP 19477 sim- _{ICMP19477 carrying a single crossover mutation in sim}

integrated with pK18mobsacB.

(Preetinanda Panda, 2014; Doctoral thesis)

Chl, Km

PBR1692 Wild Type (Duarte et al., 2004) N/A

E. coli strains

ESS β-lactam supersensitive indicator strain (Bainton et al., 1992) N/A

S17-1λpir (mTn5-gus-pgfp21) S17-1λpir carrying the Tn5 ‘cassette’ mTn5-gus-pgfp21 in

pUT. Provides conjugal function in bacterial crosses

(Sandra Vinovsky, private communication)

Amp, Km

TOP10 F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80ΔLacZΔ M15 Δ lacX74

recA1 araD139 Δ(araleu) 7697 galU galK rspL (StrR_{) endA1}

nupG

Life Technologies Str

HH26 pNJ500 Strain used for mobilisation in conjugal transfer (Grinter, 1983) Tet

Chromobacterium strains

C. violacein CV026 Mini-Tn5 transposon mutant negative for violacein-

pigment production. Biosenser strain for N-acyl homoserine lactone production.

(Latifi et al., 1995) Km

Plasmids

pTRB32oriT (pPF259) Derivative of pQE80-L (Expression vector for native or N-

terminal hexahistidine proteins), containing an origin of transfer RP4 oriT.

(M. McNeil, unpublished) Chl

pTRB32oriT::carR pTRB32oriT containing a 738 bp fragment, encompassing

KCO_04647.

This study Chl

pTRB32oriT::slyA pTRB32oriT containing a 436 bp fragment, encompassing

of KCO_21137.

This study Chl

pK18mobsacB Vector used for the delivery of knockout gene constructs (Schäfer et al., 1994) Km

pFAJ1819 pUT derivative, mini Tn5 transposon vector containing

mTn5-gus-pgfp21