Genomotyping

of

Pseudomonas

putida

strains

using

P.

putida

high-density

DNA

microarrays

implications

for

transcriptomics

studies

Pseudomonas

putida

is

the

only

fully

sequenced

P.

putida

strain

Thus

for

transcriptomics

and

proteomics

studies

with

other

P.

putida

strains

the

P.

putida

genomic

database

serves

as

standard

reference

The

utility

of

whole-genome

high-density

oligonucleotide

microarrays

for

transcriptomics

studies

of

other

Pseudomonas

strains

was

investigated

To

this

end

microarray

hybridizations

were

performed

with

genomic

DNAs

of

subcultures

of

P.

putida

-LRB-

-RRB-

the

type

strain

-LRB-

-RRB-

plasmid

strain

-LRB-

-RRB-

the

solvent-tolerant

P.

putida

and

several

other

Pseudomonas

strains

Depending

on

the

strain

tested

to

of

all

genetic

elements

were

identified

in

the

genomic

DNAs

The

efficacy

of

these

microarrays

to

study

cellular

function

was

determined

for

all

strains

included

in

the

study

The

vast

majority

of

genes

encoding

proteins

of

primary

metabolism

and

genes

involved

in

the

catabolism

of

aromatic

compounds

were

identified

in

the

genomic

DNA

of

strain

a

prerequisite

for

reliable

transcriptomics

analyses

The

genomotypic

comparisons

between

Pseudomonas

strains

were

used

to

construct

highly

discriminative

phylogenetic

relationships

and

were

indistinguishable

and

clustered

together

with

strain

in

a

separate

group

distinct

from

Pseudomonas

monteilii

-LRB-

-RRB-

clustered

well

with

P.

putida

strains

Introduction

The

sequencing

and

annotation

of

the

Pseudomonas

putida

genome

-LRB-

Nelson

et

al.

-RRB-

has

greatly

catalyzed

research

on

this

strain

and

other

academically

and

biotechnologically

relevant

but

non-sequenced

P.

putida

strains

A

number

of

key

scientific

advances

-LRB-

both

pure

and

applied

-RRB-

have

been

made

via

proteome

and

transcriptome

analysis

of

P.

putida

strains

-LRB-

Dominguez-Cuevas

et

al.

Hallsworth

et

al.

Morales

et

al.

Reva

et

al.

Santos

et

al.

Segura

et

al.

Volkers

et

al.

Yuste

et

al.

-RRB-

In

our

laboratories

we

have

studied

both

P.

putida

-LRB-

for

recent

publications

Hallsworth

et

al.

Martins

dos

Santos

et

al.

Nelson

et

al.

Timmis

-RRB-

and

the

solvent-tolerant

P.

putida

-LRB-

De

Bont

Hartmans

et

al.

Wery

and

De

Bont

-RRB-

Unlike

most

pseudomonads

solvent-tolerant

strains

possess

an

extraordinary

tolerance

to

a

broad

variety

of

toxic

solvents

-LRB-

for

reviews

De

Bont

Kieboom

and

De

Bont

Ramos

et

al.

-RRB-

A

large

number

of

high-added

value

bioconversions

involve

toxic

generally

apolar

products

-LRB-

aromatics

aliphatic

alcohols

epoxides

etc.

-RRB-

Especially

for

bioprocesses

involving

such

products

the

use

of

solvent-tolerant

P.

putida

strains

renders

advantages

in

terms

of

productivity

and

the

application

of

multiphase

media

for

product

recovery

-LRB-

Ramos-Gonzalez

et

al.

Rojas

et

al.

Wery

and

De

Bont

Wery

et

al.

Wierckx

et

al.

-RRB-

Despite

their

biotechnological

potential

the

genomes

of

and

other

Pseudomonas

strains

have

not

been

sequenced

So

recent

studies

of

their

metabolic

potential

have

been

limited

to

comparative

proteomics

-LRB-

Segura

et

al.

Volkers

et

al.

-RRB-

or

transcriptomics

based

on

the

database

information

for

P.

putida

The

genus

Pseudomonas

is

very

heterogeneous

-LRB-

Anzai

et

al.

-RRB-

even

strains

of

one

species

tend

to

vary

greatly

in

terms

of

both

phenotypic

-LRB-

Grimont

et

al.

-RRB-

and

genotypic

characteristics

-LRB-

Brosch

et

al.

-RRB-

Strains

belonging

to

the

species

P.

putida

can

be

categorized

into

biovar

A

and

B

the

former

grouping

-LRB-

A

-RRB-

is

the

more

heterogeneous

-LRB-

Brosch

et

al.

-RRB-

and

the

phylogenetic

and

metabolic

diversity

of

biovar

A

strains

has

yet

to

be

fully

characterized

and

industrially

exploited

Comparative

transcriptomics-based

approaches

have

played

a

pivotal

role

in

recent

investigations

of

complex

cellular

responses

of

P.

putida

strains

-LRB-

Dominguez-Cuevas

et

al.

Duque

et

al.

Yuste

et

al.

-RRB-

As

transcriptome

profiling

is

based

on

the

highly-sensitive

detection

of

DNA

cDNA

hybridization

DNA

sequence

similarity

determines

the

validity

of

these

analyses

The

inherent

heterogeneity

of

the

P.

putida

grouping

provides

additional

cause

for

concern

that

microarray

platforms

derived

from

strain

may

provide

a

valid

framework

for

the

study

of

nonsequenced

P.

putida

strains

We

therefore

carried

out

this

study

to

assess

the

utility

of

high-density

DNA

microarrays

for

transcriptomics

studies

of

DSM

-LRB-

subculture

of

-RRB-

DSM

-LRB-

subculture

of

-RRB-

DSM

-LRB-

the

P.

putida

type

strain

-RRB-

the

solvent-tolerant

and

other

nonsequenced

Pseudomonas

strains

In

addition

the

use

of

such

microarrays

to

establish

highly

discriminative

phylogenetic

relationships

between

these

strains

was

demonstrated

Materials

and

methods

Strains

cultivation

conditions

and

DNA

extraction

Single

colonies

from

each

Pseudomonas

strain

studied

-LRB-

see

Table

-RRB-

grown

on

Pseudomonas

Isolation

Agar

-LRB-

Difco

-RRB-

were

used

to

inoculate

Luria

Bertani

broth

-LRB-

LB

-RRB-

-LRB-

Sambrook

et

al.

-RRB-

in

duplicate

After

cultures

were

grown

overnight

at

°C

genomic

DNAs

were

prepared

from

ml

of

culture

-LRB-

approximately

cells/ml

-RRB-

using

the

Genomic

DNA

Kit

-LRB-

Qiagen

Germany

-RRB-

according

to

the

manufacturer

's

instructions

DNA

concentrations

were

determined

at

nm

using

spectrophotometer

-LRB-

NanoDrop

Wilmington

DE

USA

-RRB-

and

purity

was

confirmed

by

agarose

-LRB-

w/v

-RRB-

gel

electrophoresis

Table

and

designations

of

Pseudomonas

strains

used

in

this

studySpecies

and

strain

numberaSource

or

referencebOther

designationscP

putida

DSM

ATCC

NCIMB

putida

DSM

ATCC

ATCC

JCM

NCIMB

putida

et

al.

putida

DSM

ATCC

ICPB

NCTC

CCUG

LMG

putida

DSM

ICPB

JCM

NCIMB

putida

DSM

ICPB

JCM

NCIMB

monteilii

DSM

CCUG

LMG

CIP

fluorescens

DSM

ICPB

NCIMB

NCTC

CCUG

LMG

designations

used

in

this

studybDSMZ

Deutsche

Sammlung

von

Mikroorganismen

und

Zellkulturen

GermanycSubcultures

of

the

used

strains

available

in

other

culture

collections

ATCC

American

Type

Culture

Collection

ICPB

International

Collection

of

Phytopathogenic

Bacteria

USA

NCIMB

National

Collections

of

Industrial

and

Marine

Bacteria

UK

NCTC

National

Collection

of

Type

Cultures

UK

JCM

Japan

Collection

of

Microorganisms

Japan

CIP

Collection

bacteriènne

de

l'Institut

Pasteur

France

CCUG

Culture

Collection

University

Göteborg

Sweden

LMG

BCCM/LMG

Bacteria

Collection

Belgium

Preparation

of

biotin-labelled

fragmented

genomic

DNAs

Approximately

μg

of

heat-denatured

genomic

DNA

was

fragmented

using

U

DNaseI

-LRB-

Pharmacia

-RRB-

μg

DNA

at

°C

for

min

-LRB-

Wolfgang

et

al.

-RRB-

DNaseI

was

inactivated

by

immediate

heat

treatment

at

°C

for

min

followed

by

cooling

on

ice

An

aliquot

of

fragmented

DNA

was

analyzed

by

agarose

-LRB-

w

v

in

TAE

buffer

-RRB-

gel

electrophoresis

to

confirm

the

purity

and

establish

the

mean

size

of

DNA

fragments

-LRB-

bp

-RRB-

Approximately

μg

of

fragmented

DNA

was

labelled

with

biotin

according

to

the

Affymetrix

Expression

Analysis

Technical

Manual

for

Prokaryotic

Samples

Design

of

high-density

oligonucleotide

microarrays

and

hybridization

with

genomic

DNAs

High-density

oligonucleotide

microarrays

based

on

the

annotated

genome

of

P.

putida

-LRB-

NC

-RRB-

were

constructed

according

to

Affymetrix

specifications

-LRB-

manual

pdf

-RRB-

with

the

pair-wise

configuration

of

perfect

match

-LRB-

PM

-RRB-

and

mismatch

-LRB-

MM

-RRB-

oligonucleotides

per

gene

-LRB-

probe

set

-RRB-

The

microarrays

included

probe

sets

representing

annotated

genes

or

open

reading

frames

-LRB-

orfs

-RRB-

and

targeting

intergenic

regions

Hybridization

and

washing

of

the

microarrays

were

performed

on

a

GeneChip

Node

system

-LRB-

Hybridization

temperature

°C

Hybridization

oven

Fluidics

station

Affymetrix

Santa

Clara

CA

USA

-RRB-

following

the

supplier

's

instructions

Scanning

was

carried

out

by

ServiceXS

-LRB-

Leiden

The

Netherlands

-RRB-

on

a

high

resolution

Gene

Chip

Scanner

system

with

autoloader

-LRB-

Affymetrix

Santa

Clara

CA

USA

-RRB-

using

the

default

analysis

settings

of

the

manufacturer

-LRB-

filter

nm

pixel

size

μm

-RRB-

Hybridization

intensity

data

were

extracted

from

the

scanned

array

images

and

the

designations

for

presence

or

absence

were

calculated

with

Affymetrix

Microarray

Suite

-LRB-

MAS

-RRB-

software

A

one-sided

Wilcoxon

's

Signed

Rank

was

used

to

calculate

p-values

reflecting

the

statistical

significance

of

differences

between

PM

and

MM

of

a

probe

set

The

significance

levels

for

detection

calls

of

probe

sets

were

p-value

for

present

and

p-value

for

absent

Differences

between

PM

and

MM

were

considered

insignificant

and

removed

from

further

consideration

by

comparisons

of

the

discrimination

score

-LSB-

-LRB-

PM

MM

-RRB-

-LRB-

PM+MM

-RRB-

-RSB-

with

the

defined

discrimination

threshold

τ

-LRB-

discrimination

score

-RRB-

Comparisons

of

present/absent

designations

for

genes

in

the

different

strains

were

made

with

GeneSpring

version

-LRB-

Agilent

-RRB-

Replicate

assays

of

biological

duplicates

were

performed

for

P.

putida

DSM

and

The

deviation

caused

by

present/absent

designations

of

genes

in

only

one

of

both

replicates

was

for

DSM

and

for

regarding

all

probe

sets

for

putative

genes

and

intergenic

regions

-LRB-

Table

-RRB-

Table

indices

based

on

designations

of

presence

or

absence

-LRB-

stated

in

brackets

-RRB-

from

hybridization

signals

of

genomic

DNA

fragments

from

different

Pseudomonas

members

on

P.

putida

microarraysaStrainPresent

-LRB-

absent

-RRB-

in

percent

-LRB-

-RRB-

Putative

genes

and

intergenic

regions

-LRB-

probe

sets

-RRB-

Putative

genes

-LRB-

probe

sets

-RRB-

Putative

genes

with

assigned

function

-LRB-

probe

sets

-RRB-

P.

putida

DSM

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

P.

putida

DSM

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

P.

putida

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

P.

putida

DSM

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

P.

putida

DSM

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

P.

putida

DSM

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

P.

monteilii

DSM

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

P.

fluorescens

DSM

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aPresent/absent

designations

-LRB-

present

p

absent

p

τ

-RRB-

derived

from

a

decision

matrix

in

Affymetrix

MAS

bReplicate

assays

were

performed

for

P.

putida

DSM

and

and

the

values

listed

include

only

genes

that

were

designated

present

or

absent

in

both

replicates

Calculation

of

dendrograms

Simple

matching

similarity

matrix

based

on

the

present/absent

detection

calls

in

microarray

analysis

of

different

pseudomonads

-LRB-

SM

m/n

m

number

of

matching

probe

sets

n

total

number

probe

sets

-RRB-

and

the

microarray

genotyping

dendrogram

were

calculated

using

UPGMA

parameters

-LRB-

unweighted

pair

group

method

arithmetic

average

-RRB-

and

agglomerative

hierarchical

clustering

with

XLSTAT

version

-LRB-

Addinsoft

Paris

France

-RRB-

AFLP

analysis

In

this

paper

we

used

amplified

fragment

length

polymorphism

-LRB-

AFLP

-RRB-

-LRB-

Janssen

et

al.

Savelkoul

et

al.

Vos

et

al.

-RRB-

to

analyze

different

strains

of

P.

putida

a

P.

monteilii

and

a

Pseudomonas

fluorescens

AFLP

is

based

on

selective

amplification

of

restriction

fragments

from

totally

digested

genomic

DNA

AFLP

fingerprints

were

performed

by

KeyGene

-LRB-

Wageningen

The

Netherlands

-RRB-

Genomic

DNA

of

the

different

pseudomonads

was

digested

using

restriction

enzyme

combination

NlaIII

-LRB-

Westburg

Leusden

The

Netherlands

-RRB-

and

-LRB-

Fermentas

St.

Leon-Rot

Germany

-RRB-

according

to

manufacturers

instructions

Each

restriction

enzyme

was

combined

with

the

ligation

of

specific

linker

oligonucleotide

pairs

-LRB-

NlaIII

-RRB-

For

each

of

these

linker

combinations

AFLP

was

performed

using

nine

N/C

AFLP

primer

combinations

which

were

selected

using

genome

sequence

as

reference

and

software

package

REcomb

-LRB-

Keygene

-RRB-

for

prediction

analysis

These

primers

were

extended

with

a

terminal

dinucleotide

-LRB-

-RRB-

and

the

extensions

were

CA/AC

CA/CA

CA/CC

CA/GG

CA/TT

CT/TT

CT/CA

CT/CT

and

CT/TC

For

further

detail

please

refer

to

Van

den

Braak

et

al.

-LRB-

-RRB-

PCRs

were

performed

in

the

presence

of

radioactive

nucleotides

and

the

amplimers

obtained

were

separated

on

cm

polyacrylamide

slabgels

Using

phosphor-imaging

the

individual

presence/absence

in

a

total

of

markers

-LRB-

DNA

bands

-RRB-

per

strain

was

analyzed

The

total

marker

score

data

table

-LRB-

presence/absence

of

individual

DNA

bands

-RRB-

was

subjected

to

genetic

distance

analysis

using

simple

matching

similarity

matrix

-LRB-

SM

m/n

m

the

number

of

matched

scores

n

the

total

sample

size

-RRB-

consisting

of

similarity

indices

for

all

combinations

of

AFLP-banding

patterns

Simple

matching

coefficients

were

calculated

using

NTSYSpc-software

version

-LRB-

Exeter

Software

Setauket

NY

USA

-RRB-

To

visualize

the

relationship

between

the

strains

a

dendrogram

was

generated

using

Sequential

Agglomerative

Hierarchical

Nested

-LRB-

SAHN

-RRB-

cluster

analysis

with

the

use

of

UPGMA

parameters

Standard

PCR

and

sequencing

Diagnostic

PCR

of

selected

putative

orfs

-LRB-

-RRB-

that

were

called

absent

in

microarray

analyses

of

P.

putida

DSM

genomic

DNA

was

performed

using

proofreading

enzyme

mixture

High

Fidelity

PCR

master

-LRB-

Roche

Diagnostics

The

Netherlands

-RRB-

and

specific

primers

-LRB-

forward

reversed

forward

CAACGGCTAAACCTTTGCAT

reversed

-RRB-

Yielded

PCR-amplicons

of

expected

sizes

and

bp

respectively

were

sent

to

Baseclear

-LRB-

Leiden

The

Netherlands

-RRB-

for

sequencing

according

to

Sanger

et

al.

-LRB-

-RRB-

using

nested

sequencing

primers

-LRB-

-RRB-

and

the

resulting

sequences

were

used

for

sequence

alignments

Biological

function-derived

phylogenetic

analysis

The

concept

of

Clusters

of

Orthologous

Genes

-LRB-

COG

see

Tatusov

et

al.

-RRB-

was

used

during

analysis

of

the

genomic

content

of

nonsequenced

Pseudomonas

strains

by

linkage

to

primary

biological

function

-LRB-

Table

-RRB-

A

COG

consists

of

individual

proteins

or

groups

of

paralogs

from

at

least

three

lineages

and

thus

corresponds

to

an

ancient

conserved

domain

-LRB-

Tatusov

et

al.

-RRB-

In

the

NCBI-COG

database

proteins

of

putative

orfs

in

P.

putida

were

identified

as

COGs

and

were

arranged

in

functional

categories

-LRB-

see

-RRB-

Because

of

the

limited

number

of

COGs

in

the

functional

categories

-LRB-

A

-RRB-

RNA

processing

and

modification

-LRB-

B

-RRB-

chromatin

structure/dynamics

and

-LRB-

W

-RRB-

extracellular

structures

-LRB-

and

respectively

-RRB-

these

COGs

were

collectively

grouped

together

with

general

function

prediction

COGs

-LRB-

Category

R

-RRB-

under

the

designation

R

-LRB-

see

Table

-RRB-

Table

functional

designationa

of

genes

identified

in

P.

putida

genomic

DNACodebDescriptionCOGs

in

in

transport

and

-LRB-

-RRB-

-LRB-

-RRB-

HCoenzyme

transport

and

-LRB-

-RRB-

TSignal

transduction

-LRB-

-RRB-

ILipid

transport

and

-LRB-

-RRB-

NCell

-LRB-

-RRB-

GCarbohydrate

transport

and

-LRB-

-RRB-

PInorganic

ion

transport

and

-LRB-

-RRB-

UIntracellular

trafficking

and

-LRB-

-RRB-

EAmino

acid

transport

and

-LRB-

-RRB-

OPosttranslational

modification

protein

turnover

-LRB-

-RRB-

CEnergy

production

and

-LRB-

-RRB-

QSecondary

metabolites

biosynthesis

transport

and

-LRB-

-RRB-

-LRB-

-RRB-

VDefense

-LRB-

-RRB-

R

General

function

prediction

-LRB-

-RRB-

SFunction

-LRB-

-RRB-

MCell

membrane

-LRB-

-RRB-

DCell

cycle

-LRB-

-RRB-

Not

in

-LRB-

-RRB-

LReplication

recombination

and

-LRB-

-RRB-

aBased

on

the

Clusters

of

Orthologous

Groups

-LRB-

COG

-RRB-

system

also

see

Materials

and

methods

section

-RRB-

bCodes

of

functional

categories

of

COGcProteins

of

putative

orfs

in

P.

putida

can

be

found

in

COG

database

-LRB-

COG

-RRB-

Results

Genomic

DNA

hybridizations

with

P.

putida

high-density

DNA

microarrays

Total

genomic

DNA

from

P.

putida

strains

and

other

nonsequenced

Pseudomonas

members

-LRB-

see

Table

-RRB-

was

hybridized

to

custom

high-density

oligonucleotide

microarrays

Presence

or

absence

designations

for

each

probe

set

-LRB-

designed

for

specific

genes

and

intergenic

regions

-RRB-

were

calculated

by

the

Affymetrix

MAS

algorithm

from

the

significant

difference

-LRB-

see

Materials

and

methods

section

-RRB-

in

hybridization

intensities

between

the

corresponding

perfect

match

and

mismatch

oligonucleotides

-LRB-

Table

-RRB-

Absence

designation

is

a

synonym

for

the

absence

of

significant

signal

values

and

stands

for

divergent

DNA

still

coding

a

similar

biological

function

or

for

the

complete

absence

of

the

specific

DNA

Replicate

array

hybridizations

were

performed

for

DSM

DNA

and

DNA

only

For

these

DNAs

the

values

given

in

Table

represent

only

probe

sets

designated

present

or

absent

in

both

replicates

As

expected

the

DSM

DNA

yielded

an

almost

perfect

score

for

probe

sets

corresponding

to

all

genetic

elements

-LRB-

including

the

intergenic

regions

-RRB-

and

for

probe

sets

designed

for

genes

with

an

assigned

function

Strain

DSM

-LRB-

subculture

of

P.

putida

Teruko

-RRB-

is

a

TOL

plasmid

-LRB-

-RRB-

containing

variant

of

DSM

and

was

used

as

an

additional

control

for

the

accuracy

of

the

microarray

experiments

As

expected

both

strains

were

virtually

indistinguishable

in

the

array

hybridization

study

-LRB-

Table

-RRB-

Nonetheless

four

orfs

were

indicated

absent

in

DSM

These

orfs

were

found

to

be

called

absent

in

only

one

of

the

DSM

replicates

We

therefore

used

diagnostic

PCR

to

investigate

the

presence

of

two

of

them

-LRB-

-RRB-

in

DSM

the

other

two

appeared

less

important

due

to

their

limited

size

-LRB-

bp

-RRB-

In

both

cases

PCR

products

of

the

expected

sizes

-LRB-

and

bp

-RRB-

were

obtained

that

after

sequencing

proved

to

be

identical

to

the

homologs

Apart

from

DSM

the

solvent-tolerant

showed

the

highest

genomic

similarity

to

Nearly

of

-LRB-

-RRB-

genes

with

assigned

functions

in

the

genome

were

identified

in

the

genomic

DNA

of

-LRB-

Table

-RRB-

Approximately

of

-LRB-

putative

-RRB-

genes

without

an

assigned

function

and

of

the

intergenic

regions

were

found

to

be

present

in

the

-LRB-

data

not

shown

-RRB-

Microarray-based

genomotyping

A

dendrogram

was

constructed

-LRB-

Fig.

-RRB-

based

on

genomic

similarity

of

all

genetic

elements

of

in

the

tested

genomic

DNAs

except

for

the

control

strain

DSM

-LRB-

Table

-RRB-

DSM

DSM

-LRB-

not

shown

-RRB-

and

clustered

in

a

group

separate

from

the

other

P.

putida

strains

-LRB-

Fig.

-RRB-

The

nonsequenced

P.

fluorescens

-LRB-

DSM

-RRB-

that

was

included

as

an

out-group

did

not

cluster

with

any

other

strain

By

contrast

Pseudomonas

monteilii

DSM

that

was

included

as

a

non-putida

member

clustered

with

other

P.

putida

strains

suggesting

a

closer

relationship

with

the

P.

putida

taxonomic

grouping

-LRB-

Fig.

-RRB-

To

assess

the

validity

of

these

microarray-derived

phylogenetic

relationships

AFLP

DNA

fingerprinting

was

used

to

obtain

an

independent

phylogenetic

classification

of

strains

-LRB-

Janssen

et

al.

Savelkoul

et

al.

Vos

et

al.

-RRB-

The

phylogenetic

tree

constructed

following

AFLP

analyses

showed

an

identical

pattern

in

terms

of

strain

grouping

-LRB-

Fig.

-RRB-

Fig.

similarity

between

different

Pseudomonas

strains

a

High-density

microarray

genotyping

tree

based

on

absent/present

designations

generated

by

Affymetrix

MAS

algorithm

-LRB-

Table

-RRB-

using

simple

matching

similarity

coefficient

UPGMA

and

agglomerative

hierarchical

clustering

b

AFLP-genotyping

Tree

is

based

on

the

score

of

AFLP

markers

using

the

simple

matching

similarity

coefficient

and

Sahn

cluster

analysis

An

inventory

was

made

of

genes

encoding

proteins

belonging

to

COGs

-LRB-

Tatusov

et

al.

-RRB-

that

were

identified

in

the

different

genomic

DNAs

-LRB-

Fig.

-RRB-

Of

orfs

in

the

genomic

DNA

sequence

encode

proteins

that

have

been

categorized

into

classes

of

primary

biological

function

based

on

the

COG

system

-LRB-

COG

-RRB-

It

was

found

that

after

DSM

P.

putida

showed

the

highest

present

score

in

all

functional

classes

-LRB-

Fig.

-RRB-

The

present

designations

for

genes

encoding

COG

members

ranged

from

to

depending

on

their

primary

biological

function

-LRB-

Table

Fig.

-RRB-

The

unequal

distribution

of

the

present

designations

over

the

different

functional

classes

was

also

characteristic

of

other

Pseudomonas

strains

-LRB-

Fig.

-RRB-

Strikingly

all

pseudomonads

other

than

DSM

DSM

and

the

out-group

showed

a

comparable

distribution

pattern

of

identified

genes

over

the

different

COGs

Fig.

of

genes

encoding

COG

proteins

over

different

functional

categories

as

identified

in

Pseudomonas

strains

Functional

categories

were

adopted

from

the

COG

database

for

P.

putida

-LRB-

-RRB-

The

categories

of

biological

function

corresponding

to

COG

codes

are

given

in

Table

P.

putida

strains

are

well-known

for

their

broad

metabolic

potential

regarding

aromatic

compounds

-LRB-

Jimenez

et

al.

Wackett

for

reviews

-RRB-

Several

pseudomonads

including

P.

putida

have

been

reported

to

degrade

and/or

transform

a

variety

of

aromatics

Among

these

are

compounds

of

industrial

importance

and

there

is

an

interest

in

studying

these

metabolic

pathways

on

the

level

of

gene

expression

and

regulation

thereof

The

utility

of

microarrays

in

transcriptomics

studies

of

aromatics

metabolism

of

the

pseudomonads

under

investigation

was

appraised

The

presence

of

genes

of

central

and

peripheral

metabolic

routes

of

aromatics

were

chosen

based

on

reports

by

Jimenez

et

al.

-LRB-

-RRB-

and

Wackett

-LRB-

-RRB-

-LRB-

Table

-RRB-

All

genes

-LRB-

-RRB-

were

detected

in

DSM

and

in

DSM

Higher

divergence

was

found

for

P.

putida

DSM

-LRB-

detectable

-RRB-

DSM

and

DSM

-LRB-

both

detectable

-RRB-

and

DSM

-LRB-

detectable

-RRB-

DSM

again

behaved

as

an

out-group

with

only

of

the

genes

detectable

In

the

P.

putida

strains

used

in

the

present

study

and

DSM

almost

all

genes

for

degradation

of

benzoate

-LRB-

ben

-RRB-

homogentisate

-LRB-

hmg

mai

-RRB-

phenylalanine/tyrosine

-LRB-

phh

tyrB

-RRB-

and

catAB

were

detected

In

DSM

and

putative

regulatory

genes

of

different

pathways

-LRB-

e.g.

catR

pcaQ

pobR

phaNM

-RRB-

and

some

isoenzymes

-LRB-

e.g.

-RRB-

were

not

found

-LRB-

Table

-RRB-

Interestingly

in

all

these

strains

the

muconolactone

isomerase

-LRB-

catC

-RRB-

and

in

the

catabolism

of

phenylpropenoid

compounds

vanillin

dehydrogenase

-LRB-

vdh

-RRB-

putative

conifer

aldehyde

dehydrogenase

-LRB-

calB

except

DSM

-RRB-

and

feruloyl-CoA

synthetase

-LRB-

fcs

except

for

DSM

-RRB-

were

not

identified

and

can

thus

not

be

studied

in

microarray-based

transcriptome

analysis

Table

of

the

presence/absence

designations

of

genes

encoding

metabolic

pathways

of

aromatic

compounds

in

different

pseudomonads

derived

from

Affymetrix

decision

matrix

MAS

StrainPresent

-LRB-

-RRB-

aGenes

of

aromatic

pathways

not

identified

in

hybridizations

with

genomic

DNAP

putida

DSM

P.

putida

DSM

P.

putida

P.

putida

DSM

catC

fcs

pcaQ

phaDHMN

vdh

calB

putida

DSM

catCR

ech

fcs

pcaDQ

phaGHMN

pobAR

vdh

putida

DSM

catCR

ech

pcaBCQ

phaDHJMN

phhR

pobR

vdh

calB

monteilii

DSM

catCR

ech

fcs

pcaBDQ

phaEGHMN

pobAR

vdh

calB

fluorescens

DSM

aat

acdA

catABCR

fcs

hpd

pcaCDHIJKQT

pcm

phaAB-EG-ILMN

phhBR

pobAR

vanAB

vdh

calB

ferR

genes

-LRB-

-RRB-

involved

in

catabolism

of

aromatic

compounds

annotated

in

P.

putida

DSM

protocatechuate

-LRB-

pcaBCDFGHIJKQRT

-RRB-

catechol

branches

β-ketoadipate

pathway

-LRB-

-RRB-

homogentisate

pathway

-LRB-

hmgA

mai

-RRB-

central

meta-cleavage

-LRB-

pcm

-RRB-

p-hydroxybenzoate

-LRB-

pobAR

-RRB-

benzoate

-LRB-

-RRB-

phenylacetate

pathway

-LRB-

-RRB-

n-phenylalkanoic

acids

-LRB-

-RRB-

phenylpropenoid

compounds

-LRB-

vanAB

vdh

calB

ferR

fcs

ech

aat

acdA

-RRB-

and

phenylalanine/tyrosine

-LRB-

phhABR

hpd

pcm

-RRB-

Discussion

A

major

advantage

of

microarray-based

comparisons

of

species

is

the

ability

to

pinpoint

differences

in

individual

genes

and

intergenic

regions

Through

these

comparisons

detailed

insight

was

gained

in

the

utility

of

P.

putida

microarrays

in

transcriptomics

studies

of

different

pseudomonads

at

the

level

of

specific

categories

of

biological

function

It

was

found

that

genes

involved

in

e.g.

translation

and

nucleotide

transport

and

metabolism

could

be

identified

at

a

high

frequency

-LRB-

-RRB-

in

all

P.

putida

strains

tested

in

contrast

to

other

functional

groups

where

the

frequency

of

gene

identification

dropped

below

Whereas

P.

fluorescens

DSM

behaved

as

a

typical

out-group

in

these

functional

studies

it

was

clear

that

depending

on

the

functional

category

up

to

of

the

genes

of

this

strain

could

still

be

identified

There

is

a

biotechnological

requirement

for

P.

putida

biocatalysts

that

can

function

at

high

solvent

concentrations

such

as

strain

and

that

can

be

swiftly

optimized

for

different

bio-based

production

processes

In

our

group

studies

have

focused

on

the

construction

of

strains

that

are

able

to

convert

renewable

substrates

such

as

sugars

into

aromatics

of

interest

via

central

metabolites

-LRB-

Nijkamp

et

al.

Wierckx

et

al.

-RRB-

These

conversions

take

place

via

multiple

metabolic

pathways

each

consisting

of

several

enzymatic

steps

with

regulatory

mechanisms

that

are

being

investigated

using

as

a

model

system

A

comparative

transcriptomics

approach

is

invaluable

to

gain

detailed

insights

into

the

complex

cellular

systems

of

the

metabolically

versatile

pseudomonads

The

employment

of

the

high-density

microarrays

would

enable

highly

sensitive

and

reproducible

transcriptome

analyses

that

are

compatible

with

those

used

for

model

species

such

as

P.

aeruginosa

-LRB-

Ochsner

et

al.

Wagner

et

al.

Whiteley

et

al.

-RRB-

and

Escherichia

coli

-LRB-

Woo

et

al.

-RRB-

In

the

present

study

we

showed

that

the

use

of

microarrays

would

enable

reliable

transcriptomics

analysis

of

P.

putida

Significantly

we

found

that

of

all

pseudomonads

tested

the

genomic

content

of

P.

putida

showed

the

highest

similarity

to

that

of

P.

putida

DSM

-LRB-

-RRB-

The

vast

majority

of

genes

coding

for

proteins

involved

in

primary

metabolism

including

biosynthesis

of

important

intermediates

such

as

amino

acids

and

the

genes

involved

in

the

conversion

of

aromatic

compounds

were

shown

to

be

sufficiently

similar

to

those

of

The

high

resolution

achieved

by

comparative

genomotyping

enabled

the

identification

of

minute

genotypic

differences

between

tested

strains

making

a

meaningful

analysis

of

phylogenetic

relationships

feasible

For

example

the

genomic

DNA

of

P.

putida

DSM

was

shown

to

be

virtually

identical

to

that

of

the

control

strain

DSM

and

this

is

consistent

with

the

origin

of

strain

as

a

derivative

of

strain

-LRB-

Regenhardt

et

al.

-RRB-

The

relationship

between

the

P.

putida

DSM

and

has

been

an

issue

of

controversy

Based

on

rRNA

gene

comparisons

-LRB-

identity

-RRB-

both

strains

appeared

closely

related

however

a

DNA

DNA

hybridization

experiment

indicated

only

genome

relatedness

between

both

strains

-LRB-

Regenhardt

et

al.

-RRB-

The

results

lead

to

doubts

about

the

classification

of

both

strains

as

part

of

the

same

Pseudomonas

species

given

the

widely

accepted

recommendation

that

strains

of

the

same

species

shall

have

genome

similarities

higher

than

-LRB-

Stackebrandt

and

Goebel

-RRB-

In

the

same

study

an

appreciable

distance

between

DSM

and

was

established

by

REP-PCR

genomic

fingerprint

patterns

and

Biolog

GN

metabolic

profiling

In

our

genomotyping

approach

of

the

genes

with

an

assigned

function

of

all

genetic

elements

-LRB-

including

intergenic

regions

-RRB-

and

of

the

intergenic

regions

-LRB-

not

shown

-RRB-

were

identified

in

the

genomic

DNA

of

P.

putida

DSM

These

differences

which

are

supported

by

the

AFLP

analysis

indicate

a

considerable

phylogenetic

distance

between

DSM

and

The

diversity

within

the

species

P.

putida

was

previously

reflected

in

studies

concerning

genomic

DNA

ribotyping

-LRB-

Brosch

et

al.

-RRB-

whole

cell

protein

electrophoretic

fingerprinting

-LRB-

Vacanneyt

et

al.

-RRB-

and

Biolog/Biotype

experiments

-LRB-

Grimont

et

al.

-RRB-

In

our

genomotyping

studies

strains

of

P.

monteilii

and

P.

fluorescens

were

included

as

out-groups

P.

fluorescens

DSM

was

shown

to

be

distantly

related

to

the

other

Pseudomonas

members

tested

which

supports

its

classification

as

a

separate

species

However

P.

monteilii

DSM

clustered

well

with

P.

putida

DSM

and

to

a

lesser

extent

with

DSM

and

P.

putida

DSM

AFLP

analysis

showed

a

comparable

result

and

confirmed

the

close

relation

between

P.

putida

and

P.

monteilii

The

present

study

thus

suggests

that

DSM

should

more

accurately

be

classified

as

a

P.

putida

Other

studies

based

on

classification

of

P.

monteilii

by

classical

well

established

taxonomic

methods

do

not

support

our

findings

DNA

DNA

hybridizations

among

P.

monteilii

CFML

DSM

and

DSM

generated

relative

bindings

of

genomic

DNA

of

and

with

ΔTm

values

of

and

respectively

-LRB-

Elomari

et

al.

-RRB-

Pyoverdine

typing

-LRB-

siderotyping

-RRB-

analyzing

the

excreted

siderophores

during

iron

starvation

of

P.

monteilii

CFML

and

DSM

produced

different

patterns

for

both

strains

-LRB-

Dabboussi

et

al.

-RRB-

Phenotypically

however

P.

monteilii

was

previously

shown

to

be

highly

similar

to

P.

putida

and

could

only

be

differentiated

by

assimilation

experiments

of

the

substrates

inositol

α-aminobutyrate

and

o

m-hydroxybenzoate

-LRB-

Dabboussi

et

al.

Elomari

et

al.

-RRB-

In

conclusion

the

genomotyping

of

different

pseudomonads

using

DNA

microarrays

yielded

novel

insights

in

their

phylogenetic

relationships

and

the

underlying

identification

of

genes

and

their

distribution

over

different

primary

and

secondary

biological

functions

This

revealed

the

utility

of

microarrays

in

transcriptomics

and

classification

studies

of

these

strains

B O B I O O O O O O O O O O O B O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O

pseudomonas putida
transcriptomics
genomotyping


Calcaneal

nonunion

three

cases

and

a

review

of

the

literature

The

long-term

follow-up

of

intra-articular

calcaneal

fractures

is

often

accompanied

by

complications

Frequently

occurring

are

arthrosis

arthrofibrosis

of

the

subtalar

joint

and

malunion

Uncommon

is

the

calcaneal

nonunion

A

total

of

three

cases

is

presented

in

this

report

including

a

review

of

the

literature

The

occurrence

of

a

nonunion

appears

to

be

more

common

after

conservative

treatment

but

the

pathophysiology

remains

unclear

however

smoking

may

play

a

role

Introduction

During

long-term

follow-up

after

intra-articular

calcaneal

fractures

complications

frequently

occur

In

clinical

practice

arthrosis

and

arthrofibrosis

of

the

subtalar

joint

malunion

and

nonunion

are

encountered

-LSB-

-RSB-

Nonunion

is

only

rarely

found

and

studies

concerning

complications

after

intra-articular

calcaneal

fractures

do

not

describe

this

complication

-LSB-

-RSB-

In

only

five

studies

including

nine

patients

reports

on

nonunion

are

made

-LSB-

-RSB-

Three

patients

with

a

pseudarthrosis

after

an

intra-articular

fracture

of

the

calcaneus

are

presented

in

the

current

study

Cases

Case

A

male

adult

-LRB-

age

-RRB-

fell

from

a

height

of

m

fracturing

his

left

calcaneus

According

to

the

Essex

Lopresti

classification

he

sustained

a

joint-depression

type

and

a

type

IIA

fracture

according

to

the

Sanders

classification

There

was

no

injury

to

the

soft

tissues

After

days

the

fracture

was

operated

on

using

percutaneous

reduction

and

fixation

according

to

the

technique

of

Forgon

and

Zadravecz

-LSB-

-RSB-

Post-operative

treatment

consisted

of

active

range

of

motion

exercises

and

months

non-weight

bearing

Initial

recovery

was

uncomplicated

Two

years

after

the

trauma

the

patient

however

returned

to

our

outpatient

department

with

complaints

of

painful

walking

and

unable

to

work

A

computed

tomography

-LRB-

CT

-RRB-

scan

showed

a

pseudarthrosis

of

the

calcaneus

and

talocalcaneal

joint

surface

incongruence

-LRB-

Fig.

-RRB-

An

injection

of

cc

lidocain

in

the

subtalar

joint

temporarily

reduced

pain

A

subtalar

joint

arthrodesis

was

performed

fusing

the

talus

and

calcaneus

with

the

use

of

bone

graft

gained

from

the

iliac

crest

After

year

the

arthrodesis

has

fully

consolidated

the

patient

is

pain

free

but

has

currently

not

yet

returned

to

work

Fig.

Case

Axial

CT-scan

image

months

after

percutaneous

reduction

and

fixation

showing

nonunion

b

Sagittal

view

Case

A

female

patient

aged

sustained

a

fracture

of

the

right

calcaneus

after

a

fall

from

the

stairs

-LRB-

Essex

Lopresti

joint

depression

type

Sanders

type

IIB

-RRB-

As

patient

was

seen

three

weeks

after

the

trauma

the

initial

treatment

was

conservatively

consisting

of

week

of

plaster-of-Paris

and

months

non-weight

bearing

with

active

range

of

motion

exercises

Two

months

later

the

patient

was

unable

to

bear

weight

at

the

fractured

side

due

to

pain

A

CT

showed

a

delayed

union

of

the

calcaneus

-LRB-

Fig.

-RRB-

Peroperatively

months

after

trauma

there

was

a

nonunion

of

the

fracture

without

apparent

signs

of

arthrosis

of

the

talocalcaneal

joint

surface

Because

of

the

smoking

habits

and

diabetes

in

this

patient

as

independent

causes

of

high

wound

complication

rates

an

early

subtalar

arthrodesis

with

bone

graft

harvested

from

the

tibial

tuberosity

was

performed

instead

of

a

correcting

osteotomy

Three

months

after

the

salvage

operation

the

arthrodesis

had

consolidated

the

patient

is

walking

pain

free

Fig.

Case

Semi-coronal

CT-scan

image

months

after

conservative

treatment

showing

delayed

union

b

Sagittal

view

Case

A

male

adult

of

sustained

a

bilateral

calcaneal

fracture

after

jumping

of

a

first

storey

balcony

The

radiographs

showed

a

comminuted

intra-articular

calcaneal

fracture

according

to

the

Essex

Lopresti

classification

at

both

sides

and

Sanders

type

IIC

and

IIIAC

fractures

on

the

left

and

right

foot

respectively

Initial

treatment

was

conservative

consisting

of

week

of

plaster-of-Paris

and

months

non-weight

bearing

with

active

range

of

motion

exercises

Thirteen

months

later

the

patient

returned

to

the

outpatient

department

with

complaints

of

pain

at

the

left

heel

Additional

radiographs

and

CT

showed

a

nonunion

of

the

fracture

of

the

left

calcaneus

-LRB-

Fig.

-RRB-

A

subtalar

arthrodesis

was

performed

with

bone

graft

from

the

tibial

tuberosity

Two

cannulated

screws

were

inserted

from

the

tuberosity

of

the

calcaneus

bridging

the

primary

fracture

line

The

after

treatment

was

complicated

with

a

superficial

infection

which

could

be

treated

adequately

with

intravenous

antibiotics

Six

months

after

the

secondary

fusion

the

patient

was

able

to

walk

pain

free

Fig.

Case

Semi-coronal

CT-scan

image

months

after

conservative

treatment

showing

nonunion

b

Sagittal

view

Discussion

Improper

healing

after

an

intra-articular

calcaneal

fracture

carries

a

high

morbidity

Three

groups

A

malunion

B

nonunion

and

C

osteonecrosis

were

suggested

by

Zwipp

-LSB-

-RSB-

These

groups

are

further

divided

according

to

the

presence

of

joint

incongruence

varus/valgus

loss

of

height

translation

of

bones

and

luxation

Treatment

of

these

complications

after

intra-articular

calcaneal

fractures

depends

upon

the

presence

of

these

conditions

-LSB-

-RSB-

The

first

written

report

on

calcaneal

fracture

nonunion

was

by

Thomas

-LRB-

-RRB-

He

presented

a

female

patient

treated

non-operatively

who

developed

a

calcaneal

nonunion

after

months

-LSB-

-RSB-

A

correction

of

the

displaced

fragment

was

performed

and

stabilized

using

plate

osteosynthesis

and

bone

graft

The

patient

was

able

to

fully

bear

weight

weeks

after

this

procedure

Gehr

described

a

male

patient

with

an

intra-articular

comminuted

fracture

of

the

calcaneus

-LSB-

-RSB-

This

fracture

was

treated

with

open

reduction

and

internal

plate

osteosynthesis

After

removal

of

the

plate

at

months

the

patient

returned

to

the

clinic

with

local

swelling

and

pain

during

walking

A

nonunion

was

seen

and

a

correction

osteotomy

was

performed

with

bone

graft

and

screw

fixation

The

fracture

showed

healing

at

weeks

Karakurt

et

al.

-LSB-

-RSB-

presented

one

male

patient

with

a

nonunion

of

the

calcaneus

after

conservative

treatment

The

patient

sustained

a

comminuted

open

calcaneal

fracture

which

was

treated

in

plastercast

for

months

Eight

months

after

trauma

the

patient

was

unable

to

walk

without

crutches

because

of

severe

pain

of

the

heel

After

removing

fibrotic

tissue

the

calcaneus

was

filled

with

bone

graft

Eight

months

after

the

operation

patient

was

able

to

walk

and

work

without

pain

Zwipp

and

Rammelt

-LSB-

-RSB-

reported

two

patients

with

a

nonunion

of

the

calcaneus

A

female

patient

with

a

Sanders

IIC

fracture

with

a

luxation

of

the

tuberosity

fragment

was

treated

conservatively

An

arthrodesis

of

the

subtalar

and

calcaneocuboid

joint

was

performed

months

after

trauma

Postoperatively

the

American

Orthopaedic

foot

and

Ankle

Society

hindfoot

score

improved

significantly

compared

with

pre-operative

values

The

second

patient

was

a

female

polytrauma

patient

After

percutaneous

reduction

and

fixation

she

was

diagnosed

with

a

nonunion

year

after

the

trauma

for

which

a

subtalar

joint

arthrodesis

was

performed

No

data

on

outcome

was

provided

in

this

case

From

cases

described

in

literature

and

in

the

current

report

no

similarities

that

could

indicate

a

risk

factor

of

nonunion

could

be

found

There

are

no

apparent

similarities

besides

the

nonunion

-LRB-

Table

-RRB-

There

might

be

a

trend

of

nonunion

occurring

after

conservative

treatment

suggesting

that

less

rigid

or

no

fixation

may

play

a

role

in

causing

fracture

nonunion

in

the

calcaneus

However

Howard

et

al.

-LSB-

-RSB-

saw

no

cases

of

nonunion

in

a

group

of

intra-articular

calcaneal

fractures

treated

nonoperative

Patient

age

varied

between

and

both

sexes

were

affected

equally

often

and

initial

treatment

differed

between

patients

Karakurt

et

al.

-LSB-

-RSB-

suggested

that

smoking

could

be

the

cause

of

the

nonunion

All

patients

in

this

report

were

smokers

For

tibial

fractures

strong

evidence

exists

for

delayed

fracture

healing

in

smokers

-LSB-

-RSB-

And

a

significant

lower

union-rate

was

seen

after

subtalar

arthrodesis

in

smokers

versus

non-smokers

-LSB-

-RSB-

Assous

et

al.

-LSB-

-RSB-

however

saw

no

difference

in

fracture

healing

between

smokers

and

non-smokers

in

a

small

series

of

intra-articular

calcaneal

fractures

treated

operatively

Table

of

patients

presented

in

the

literature

after

a

calcaneal

nonunionStudyGenderAgeInitial

treatmentSalvage

procedureFollow-up

-LRB-

months

-RRB-

Thomas

and

Wilson

-LSB-

-RSB-

plate

bone

et

al.

-LSB-

-RSB-

Subtalar

et

al.

-LSB-

-RSB-

screws

bone

et

al.

-LSB-

-RSB-

and

Rammelt

-LSB-

-RSB-

arthrodesis

calcaneocuboid

joint

fusion

arthrodesis

Current

The

follow-up

period

after

the

salvage

procedure

is

given

In

contrast

to

the

infrequent

occurrence

of

the

calcaneal

nonunion

stated

in

the

case

reports

above

Thermann

et

al.

-LSB-

-RSB-

describe

an

in

incidence

of

nonunion

-LRB-

n

-RRB-

in

a

group

of

patients

receiving

a

subtalar

arthrodesis

for

persisting

invalidating

pain

after

an

intra-articular

calcaneal

fracture

In

these

four

patients

the

nonunion

coincided

with

painful

subtalar

posttraumatic

arthrosis

delineating

the

indication

for

a

subtalar

arthrodesis

instead

of

a

correction

osteotomy

with

internal

fixation

Calcaneal

nonunion

has

been

reported

on

infrequently

in

the

literature

but

the

number

of

patients

seen

by

Thermann

and

our

own

series

suggests

that

the

incidence

might

be

higher

than

expected

O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B B O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

calcaneus fracture pseudarthrosis nonunion