Comparative

genomics

of

Streptomyces

avermitilis

Streptomyces

cattleya

Streptomyces

maritimus

and

Kitasatospora

aureofaciens

using

a

Streptomyces

coelicolor

microarray

system

DNA/DNA

microarray

hybridization

was

used

to

compare

the

genome

content

of

Streptomyces

avermitilis

Streptomyces

cattleya

Streptomyces

maritimus

and

Kitasatospora

aureofaciens

with

that

of

Streptomyces

coelicolor

-LRB-

-RRB-

The

array

data

showed

an

about

agreement

with

the

genome

sequence

data

available

for

S.

avermitilis

and

also

showed

a

number

of

trends

in

the

genome

structure

for

Streptomyces

and

closely

related

Kitasatospora

A

core

central

region

was

well

conserved

which

might

be

predicted

from

previous

research

and

this

was

linked

to

a

low

degree

of

gene

conservation

in

the

terminal

regions

of

the

linear

chromosome

across

all

four

species

Between

these

regions

there

are

two

areas

of

intermediate

gene

conservation

by

microarray

analysis

where

gene

synteny

is

still

detectable

in

S.

avermitilis

Nonetheless

a

range

of

conserved

genes

could

be

identified

within

the

terminal

regions

Variation

in

the

genes

involved

in

differentiation

transcription

DNA

replication

etc.

provides

interesting

insights

into

which

genes

in

these

categories

are

generally

conserved

and

which

are

not

The

results

also

provide

target

priorities

for

possible

gene

knockouts

in

a

group

of

bacteria

with

a

very

large

numbers

of

genes

with

unknown

functions

compared

to

most

bacterial

species

Introduction

Streptomyces

are

a

group

of

aerobic

high

G+C

Gram

positive

bacteria

that

undergo

complex

differentiation

to

form

filamentous

mycelium

aerial

hyphae

and

spores

In

addition

they

produce

a

broad

range

of

secondary

metabolites

including

antibiotics

antiparasitic

agents

herbicides

anti-cancer

drugs

and

various

enzymes

of

industrial

importance

Two

Streptomyces

species

have

had

their

complete

genome

sequences

published

namely

the

model

organism

Streptomyces

coelicolor

-LRB-

G+C

-RRB-

and

avermictin

producer

Streptomyces

avermitilis

-LRB-

G+C

-RRB-

-LRB-

Bentley

et

al.

Ikeda

et

al.

-RRB-

Two

important

aspects

of

the

genomes

structures

of

Streptomyces

were

supported

by

sequence

data

Firstly

that

the

genome

size

of

Streptomyces

is

large

compared

to

other

bacteria

basepairs

for

S.

coelicolor

-LRB-

protein

coding

genes

-RRB-

and

bp

-LRB-

protein

coding

genes

-RRB-

for

S.

avermitilis

Secondly

that

the

genomes

of

these

two

species

are

linear

and

both

ends

contain

unique

terminal

inverted

repeats

that

probably

covalently

bind

a

terminal

protein

Terminal

inverted

repeats

and

covalently

bound

terminal

proteins

are

not

found

in

the

limited

number

of

other

bacteria

that

have

linear

chromosomes

such

as

Borrelia

burgdorferi

and

Agrobacterium

tumefaciens

and

up

to

the

present

seem

to

be

unique

to

the

Streptomyces

and

perhaps

other

Actinobacteria

-LRB-

Lin

et

al.

Chen

et

al.

Goodner

et

al.

Huang

et

al.

-RRB-

Over

Streptomyces

strains

are

present

in

the

Ribosomal

Database

Project

-LRB-

-RRB-

over

are

available

at

the

American

Type

Culture

Collection

-LRB-

-RRB-

and

many

more

are

held

in

both

public

and

private

culture

collections

throughout

the

world

Analysis

of

the

small

subunit

ribosomal

RNA

gene

sequences

of

Streptomyces

confirms

that

they

form

a

monophyletic

clade

but

one

with

considerable

diversity

In

addition

there

is

significant

gene

diversity

at

the

interspecies

level

across

the

genomes

of

both

completely

sequenced

Streptomyces

with

gene

unique

to

S.

avermitilis

and

genes

unique

to

S.

coelicolor

This

makes

them

particularly

interesting

targets

for

comparative

genomic

studies

In

this

study

we

chose

four

species

to

begin

an

analysis

of

the

genomic

diversity

of

the

Streptomyces

S.

avermitilis

was

chosen

because

of

the

availability

of

the

complete

genome

sequence

of

this

species

while

Streptomyces

maritimus

was

chosen

because

of

its

intermediate

position

in

terms

of

phylogeny

within

the

Streptomyces

Streptomyces

cattleya

was

chosen

because

based

on

small

subunit

ribosomal

RNA

sequence

this

species

is

phylogenetically

quite

divergent

from

S.

coelicolor

and

branches

near

the

root

of

the

Streptomyces

clade

Streptomyces

cattleya

is

a

β-lactam

producing

species

Finally

Kitasatospora

aureofaciens

was

chosen

as

this

genus

is

very

closely

related

to

the

Streptomyces

The

availability

of

two

microarrays

for

S.

coelicolor

-LRB-

Lum

et

al.

Huang

et

al.

Vinciotti

et

al.

-RRB-

makes

possible

a

comparative

genomic

analysis

of

Streptomyces

species

The

genes

that

make

up

the

genome

of

S.

coelicolor

have

been

classified

based

on

scheme

of

Riley

and

colleagues

for

E.

coli

and

modified

for

S.

coelicolor

-LRB-

-RRB-

A

microarray

analysis

of

the

genomes

of

these

Streptomyces

using

the

S.

coelicolor

microarray

is

able

to

provide

a

wide

ranging

comparative

analysis

of

the

conserved

genome

content

of

these

Streptomyces

This

type

of

approach

where

a

heterologous

microarray

is

used

to

analyze

the

genome

content

of

a

range

of

strains

or

species

has

been

successfully

used

in

a

wide

range

of

organisms

-LRB-

Akman

and

Aksoy

Akman

et

al.

Behr

et

al.

Chan

et

al.

Cho

and

Tiedje

Dorrell

et

al.

Dziejman

et

al.

Fitzgerald

et

al.

Gill

et

al.

Leonard

et

al.

Murray

et

al.

Porwollik

et

al.

Salama

et

al.

Israel

et

al.

Rajashekara

et

al.

-RRB-

The

strains

analyzed

using

this

approach

range

from

intraspecies

comparisons

such

as

Campylobacter

jejuni

Vibrio

cholerae

and

Staphylococcus

aureus

-LRB-

Dorrell

et

al.

Dziejman

et

al.

Fitzgerald

et

al.

-RRB-

to

interspecies

comparisons

such

as

Sodalis

glossinidiusversus

an

Escherichia

coli

array

Salmonella

bongori

versus

a

Salmonella

enterica

array

Shewanella

species

versus

Shewanella

oneidensis

and

E.

coli

arrays

and

Brucella

species

versus

a

Brucella

melitensis

array

-LRB-

Akman

et

al.

Chan

et

al.

Murray

et

al.

Rajashekara

et

al.

-RRB-

In

this

study

we

used

both

versions

of

the

S.

coelicolor

genome

microarrays

to

compare

the

gene

complements

of

the

three

Streptomyces

species

and

one

Kitasatospora

species

The

genus

Kitasatospora

is

closely

related

to

the

genus

Streptomyces

in

terms

of

morphology

chemical

taxonomy

and

small

subunit

ribosomal

RNA

sequence

analysis

Thus

the

choice

of

a

species

from

this

genus

acts

as

potential

outgroup

in

terms

of

overall

genome

structure

In

terms

of

genes

that

are

conserved

the

types

of

genes

of

particular

interest

include

genes

involved

in

secondary

metabolism

genes

involved

in

chromosome

replication

genes

in

the

terminal

regions

of

the

chromosome

sigma

factors

genes

involved

in

differentiation

and

hypothetical

genes

In

terms

of

gene

absence

the

distribution

of

such

genes

along

the

chromosome

and

the

apparent

absence

of

any

major

housekeeping

genes

in

a

specific

species

are

of

interest

This

information

provides

insights

into

genes

that

make

up

the

core

complement

for

a

member

of

the

Streptomyces

and

into

which

genes

are

central

to

defining

a

Streptomyces

species

Materials

and

methods

phylogeny

This

was

carried

out

on

selected

small

subunit

ribosomal

RNA

gene

sequences

obtained

from

Ribosomal

Database

Project-II

Release

-LRB-

-RRB-

and

aligned

using

CLUSTALX

-LRB-

Thompson

et

al.

-RRB-

The

analysis

was

carried

out

using

Neighbor-Joining

algorithm

from

the

same

program

In

the

case

of

S.

maritimus

the

taxonomy

of

the

strain

was

confirmed

by

DNA

sequencing

of

the

ribosomal

RNA

gene

Arrays

Two

series

of

arrays

that

cover

about

of

the

complete

genome

of

Streptomyces

coelicolor

-LRB-

-RRB-

-LRB-

Lum

et

al.

-RRB-

were

used

in

this

study

Both

arrays

are

PCR

arrays

but

from

different

sources

namely

Stanford

University

USA

and

the

University

of

Surrey

UK

and

made

up

of

different

PCR

products

The

Stanford

array

as

used

in

this

study

contained

sequences

covering

open

reading

frames

The

Surrey

microarray

is

made

up

of

unique

PCR

amplified

sequences

from

the

chromosome

and

from

There

are

an

additional

non-unique

alternative

and

cross-hybridizing

sequences

that

are

also

spotted

on

to

the

array

together

with

no

probe

spots

and

control

spots

The

two

types

of

arrays

were

used

to

improve

validation

with

a

system

using

heterologous

hybridization

however

only

the

University

of

Surrey

array

was

hybridized

and

analyzed

in

duplicate

The

major

difference

between

the

two

arrays

was

that

the

Surrey

array

did

not

include

a

number

of

transposition

element

related

genes

although

there

were

other

overlap

differences

The

sequences

of

the

PCR

products

are

not

available

for

either

array

due

to

intellectual

property

protection

requirements

Strains

and

growth

conditions

S.

coelicolor

-LRB-

-RRB-

-LRB-

-RRB-

S.

avermitilis

ATCC

S.

cattleya

ATCC

S.

maritimus

Yang-Ming

and

K.

aureofaciens

ATCC

were

used

in

these

studies

Fresh

spores

were

collected

and

mycelium

cultured

in

TSB

liquid

medium

with

glycine

at

°C

overnight

Preparation

of

labeled

DNA

Genomic

DNA

from

a

stationary

phase

culture

was

purified

by

the

salting

out

procedure

-LRB-

Pospiech

and

Neumann

-RRB-

and

had

been

sonicated

to

Kb

Four

to

six

micrograms

of

sonicated

genomic

DNA

were

used

as

template

and

this

was

denatured

in

the

presence

of

μg

of

GC-content

random

hexamers

in

a

total

volume

of

μl

at

°C

for

min

The

mixture

was

then

snap-cooled

on

ice

before

adding

the

remaining

reaction

components

μl

of

or

-LRB-

Amersham

Pharmacia

Biotech

-RRB-

Klenow

fragment

-LRB-

NEB

-RRB-

Klenow

buffer

μl

dNTP

-LRB-

mM

dATP

mM

dTTP

mM

dGTP

and

mM

dCTP

-RRB-

and

μl

The

random

primed

labeling

reaction

was

carried

out

for

h

at

°C

Buffer

exchange

purification

and

concentration

of

the

DNA

products

was

accomplished

by

three

cycles

of

diluting

the

reaction

mixture

in

ml

TE

buffer

-LRB-

mM

Tris

and

mM

EDTA

pH

-RRB-

and

filtering

though

a

microconcentrators

-LRB-

Millipore

-RRB-

Microarray

hybridization

and

data

analysis

The

two

DNA

pools

to

be

compared

were

mixed

and

applied

to

an

array

in

a

hybridization

mixture

that

contained

SSC

SDS

and

μg

yeast

tRNA

-LRB-

total

μl

-RRB-

which

had

been

heated

at

°C

for

min

before

being

applied

to

array

Hybridization

took

place

under

a

glass

coverslip

sealed

by

glue

in

a

humidified

Omnislide

-LRB-

Thermo

Hybaid

-RRB-

at

°C

for

h

The

slides

were

washed

dried

and

scanned

for

fluorescence

using

a

GenePix

TM

scanner

-LRB-

Axon

instruments

-RRB-

Average

signal

intensity

and

local

background

measurements

were

obtained

for

each

spot

on

each

array

using

GenePixPro

software

The

dataset

was

screened

for

aberrant

spots

and

these

were

eliminated

from

the

analysis

after

manual

checking

Most

genes

are

present

in

duplicate

on

the

two

arrays

and

the

signal

from

each

pair

of

spots

was

inputted

into

the

computer

program

available

from

ScanAlyze

-LRB-

Eisen

et

al.

Gollub

et

al.

-RRB-

The

data

was

then

processed

into

a

mean

ratio

format

The

dataset

was

normalized

for

each

array

separately

and

outputted

to

Excel

where

after

checking

the

alignment

of

the

datasets

from

each

array

a

mean

signal

for

each

common

gene

was

calculated

Genes

that

were

absent

from

either

array

mostly

transposon

related

genes

in

the

University

of

Surrey

array

were

not

included

in

the

analysis

Based

on

Bentley

et

al.

the

mean

signal

and

standard

deviation

for

the

core

region

of

genes

from

to

was

calculated

The

standard

deviation

was

used

to

set

a

cut-off

for

gene

absence

at

below

the

core

mean

The

microarray

data

is

presented

relative

to

the

S.

coelicolor

standard

in

two

ways

This

is

either

as

a

color

plot

of

the

genes

where

green

presents

a

negative

hybridization

signal

black

represents

an

equal

hybridization

signal

and

red

indicates

a

positive

hybridization

signal

using

the

program

Treeview

-LRB-

Eisen

et

al.

-RRB-

or

as

numeric

values

for

the

signal

from

each

gene

The

microarray

data

for

the

four

species

described

here

and

additional

unpublished

species

can

be

accessed

via

rkirby@ym.edu.tw

Comparison

of

the

microarray

dataset

for

S.

avermitilis

with

the

complete

genome

sequence

The

nucleotide

sequences

for

all

the

identified

open

reading

frame

from

the

S.

avermitilis

genome

sequence

-LRB-

Ikeda

et

al.

-RRB-

were

compared

with

the

genome

sequence

of

S.

coelicolor

using

blastn

limiting

the

output

to

the

best

match

This

E

value

dataset

for

the

genes

was

then

aligned

with

the

S.

avermitilis

microarray

dataset

and

a

comparison

plotted

as

a

scatterplot

Genes

showing

disagreement

between

the

two

datasets

were

identified

based

on

a

Standard

Deviation

-LRB-

SD

-RRB-

cutoff

for

the

microarray

dataset

and

a

cutoff

for

the

blast

value

Analysis

of

gene

presence

across

the

chromosome

A

graphical

display

was

created

by

counting

the

number

of

gene

detected

as

present

from

the

signal

based

on

the

cutoff

from

each

normalized

microarray

dataset

using

a

moving

window

of

genes

in

steps

of

one

Results

and

discussion

Comparison

of

S.

avermitilis

S.

cattleya

S.

maritimus

and

K.

aureofaciens

with

the

S.

coelicolor

genome

In

total

after

spot

and

data

validation

a

total

of

open

reading

frames

were

included

in

this

analysis

as

presence

on

both

types

of

array

and

giving

analyzable

signal

on

all

three

arrays

Validity

in

this

study

was

initially

obtained

by

using

microarrays

from

two

sources

that

presumably

use

different

PCR

products

to

create

the

arrays

In

addition

the

University

of

Surrey

array

was

hybridized

and

analyzed

in

duplicate

In

terms

of

gene

absence

based

on

two

standard

deviations

as

described

in

the

Materials

and

methods

section

the

agreement

between

the

Stanford

array

and

the

duplicated

University

of

Surrey

array

was

about

while

the

agreement

between

the

two

University

of

Surrey

arrays

was

about

In

order

to

minimize

the

effect

of

divergent

individual

array

spots

the

signal

mean

for

each

gene

from

the

three

arrays

was

used

throughout

this

study

In

this

study

the

genomic

content

of

three

Streptomyces

species

and

one

Kitasatospora

species

with

divergent

taxonomy

antibiotic

production

and

SSU

rRNA

sequence

are

compared

using

two

different

S.

coelicolor

microarrays

It

is

clear

that

there

are

inherent

limitations

to

this

approach

Firstly

only

gene

absence

or

divergence

rather

than

the

presence

of

new

genes

can

be

identified

Secondly

it

is

not

possible

to

clearly

separate

the

absence

of

a

gene

from

the

presence

of

a

divergent

homologue

of

the

same

gene

Finally

although

the

order

of

the

genes

in

S.

coelicolor

and

S.

avermitilis

are

known

from

their

complete

genome

sequences

and

are

well

conserved

this

does

not

mean

that

the

synteny

of

most

of

them

is

conserved

in

other

Streptomyces

species

However

the

detection

of

synteny

across

Actinobacteria

including

Mycobacterium

tuberculosis

Corynebacteriun

glutamicum

and

other

species

-LRB-

Bentley

et

al.

and

unpublished

data

-RRB-

supports

a

conserved

central

core

structure

to

the

genomes

of

the

Actinomycetes

and

a

priori

most

Streptomyces

Thus

although

major

chromosomal

reorganizations

in

the

central

core

region

can

not

be

detected

by

microarray

data

a

basic

chromosomal

structure

can

be

assumed

as

a

first

approximation

namely

a

linear

chromosome

with

variable

terminal

regions

and

a

relatively

well

conserved

core

region

When

the

pooled

data

from

the

two

arrays

for

the

four

species

was

analyzed

using

labeled

S.

coelicolor

A

-LRB-

-RRB-

chromosomal

DNA

compared

to

heterologous

labeled

chromosomal

DNA

a

wide

range

of

signal

variation

could

be

noted

and

this

is

shown

in

Supplementary

Fig.

The

SSU

rRNA

tree

places

the

divergence

of

these

four

strains

from

S.

coelicolor

as

S.

cattleya

K.

aureofaciens

S.

maritimus

S.avermitilis

-LRB-

Fig.

-RRB-

Gene

differences

were

present

in

the

order

S.

cattleya

K.

aureofaciens

S.

avermitilis

S.

maritimus

based

on

cutoff

below

the

mean

signal

for

the

core

region

genes

The

microarray

data

thus

shows

general

agreement

with

S.

cattleya

and

K

aureofaciens

being

more

divergent

and

the

other

two

species

being

relatively

closer

It

is

interesting

to

note

that

the

Kitasatospora

species

used

in

this

study

K.

aureofaciens

shows

the

same

general

structure

as

the

Streptomyces

species

This

is

not

unexpected

and

confirms

the

close

relationship

between

Kitasatospora

and

Streptomyces

and

agrees

with

the

SSU

rRNA

tree

data.Fig

rRNA

phylogenetic

tree

of

selected

Streptomyces

species

and

other

Actinomycetes

that

have

known

complete

genome

sequences

The

species

analyzed

by

microarray

are

indicated

in

bold

Further

support

of

the

reliability

of

the

data

comes

from

a

comparison

of

the

blastn

E

values

for

all

genes

and

the

microarray

data

as

shown

in

the

Fig.

scatterplot

This

indicated

out

of

genes

show

gene

absence

by

microarray

when

they

seem

to

be

present

by

blastn

and

out

of

gene

show

gene

presence

by

microarray

when

they

seem

to

be

absent

by

blastn

these

results

are

both

based

on

cutoffs

of

for

the

microarray

data

and

for

the

E

value

This

gives

an

overall

reliability

for

S.

coelicolor

compared

to

S.

avermitilis

of

Potential

errors

factors

include

in

the

case

of

the

former

type

of

error

poor

spotting

of

the

array

at

that

point

and

choice

of

the

PCR

product

sequence

-LRB-

the

comparison

is

with

the

whole

gene

as

the

PCR

products

are

not

available

-RRB-

and

in

the

latter

case

cross-hybridization

between

multiple

gene

copies

or

a

unreliable

hybridization

signal

due

to

poor

washing

in

that

area

However

the

results

for

S.

avermitilis

clearly

support

the

reliability

of

the

genome

comparisons

produced

by

this

study.Fig

comparing

gene

presence/absence

based

on

the

microarray

data

and

gene

presence/absence

based

in

blastn

between

Streptomyces

coelicolor

and

Streptomyces

avermitilis

See

Material

and

methods

for

details

Box

A

and

Box

C

includes

genes

identified

as

absent

in

S.

avermitilis

by

the

microarray

dataset

but

present

using

blastn

and

genes

present

in

S.

avermitilis

using

blastn

but

identified

as

absent

by

the

microarray

dataset

Box

B

includes

genes

that

are

correctly

identified

as

absent

by

the

microarray

dataset

Distribution

of

gene

differences

across

the

complete

chromosome

of

S.

coelicolor

for

all

four

other

Streptomyces

species

The

whole

chromosome

microarray

dataset

supports

the

following

structure

for

the

Streptomyces

chromosome

Based

on

Fig.

and

Supplementary

Fig.

there

is

a

central

core

of

conserved

probably

syntenous

genes

that

can

be

found

across

many

Actinomycetes

and

in

the

S.

coelicolor

genome

this

reach

from

about

to

-LRB-

Bentley

et

al.

-RRB-

The

regions

between

and

and

between

and

are

also

quite

well

conserved

between

the

Streptomyces

studied

here

as

well

as

being

syntenous

between

the

S.

coelicolor

and

S.

avermitilis

genome

sequences

However

they

are

not

present

when

the

genomes

of

these

two

species

are

compared

bioinformatically

to

other

divergent

Actinomycetes

These

two

regions

seem

to

be

two

genus

specific

areas

Figure

also

clearly

shows

that

gene

conservation

drops

off

dramatically

in

the

terminal

region

The

regions

from

the

left

terminus

to

and

from

to

the

right

terminus

show

much

higher

gene

divergence

that

the

rest

of

the

chromosome

This

agrees

with

the

results

for

the

S.

ambofaciens

sequencing

studies

of

that

species

terminal

regions

-LRB-

Choulet

et

al.

b

-RRB-

The

gene

conservation

levels

averaged

across

the

four

species

are

as

follows

left

terminal

region

-LRB-

-RRB-

left

genus

specific

region

-LRB-

-RRB-

core

region

-LRB-

-RRB-

right

genus

specific

region

-LRB-

-RRB-

and

right

terminal

region

-LRB-

-RRB-

It

is

noticeable

that

neither

the

size

nor

the

distribution

of

conserved

genes

is

symmetrical

between

the

two

terminal

regions

or

the

two

genus

specific

regions

Notably

the

genus

specific

region

actually

has

a

higher

frequency

of

gene

conservation

than

the

core

regions

as

a

whole

and

that

the

left

terminal

region

is

much

larger

than

the

right

terminal

region

This

possibly

represents

horizontal

exchange

of

terminal

regions

by

recombination

between

strains/species

that

involves

only

one

terminal

region

Such

an

event

would

give

rise

to

asymmetric

gene

conservation

similar

to

that

detected

here.Fig

of

gene

presence

across

the

four

species

Created

using

a

moving

window

of

genes

and

counting

the

number

of

genes

with

a

microarray

signal

below

the

mean

for

the

core

region

genes

The

Y

axis

is

the

count

for

gene

presence

In

the

Karoonuthaisiri

et

al.

-LRB-

-RRB-

study

of

regional

gene

expression

in

S.

coelicolor

the

boundaries

for

higher

transcript

levels

during

vegetative

growth

were

placed

at

Mb

for

the

left

arm

and

Mb

for

the

right

arm

The

former

is

midway

across

the

left

genus

specific

region

and

the

latter

approximately

agrees

with

the

boundary

between

the

core

and

the

right

genus

specific

region

As

the

core

region

boundaries

are

also

defined

in

terms

of

synteny

with

the

Mycobacterium

and

Corynebacterium

genomes

as

well

as

the

data

presented

here

this

supports

the

idea

that

the

S.

coelicolor

chromosome

structure

is

asymmetrical

with

respect

to

both

gene

conservation

and

gene

function

It

should

be

noted

that

because

we

are

using

only

S.

coelicolor

as

the

source

of

the

array

data

the

results

do

not

imply

that

the

genomes

of

S.

cattleya

S.

maritimus

and

K.

aurefaciens

are

asymmetric

However

it

should

be

noted

that

the

S.

avermitilis

genome

is

also

asymmetric

-LRB-

Ikeda

et

al.

-RRB-

Notably

there

are

identifiable

regions

where

all

four

species

show

a

significant

degree

of

concurrent

gene

absence

outside

of

the

terminal

regions

-LRB-

Table

-RRB-

The

regions

of

high

gene

divergence

are

shown

in

Supplementary

Fig.

in

detail

Previously

Bentley

et

al.

identified

regions

in

the

S.

coelicolor

chromosome

that

were

potentially

laterally

acquired

regions

This

analysis

pinpoints

all

of

these

regions

and

quite

accurately

usually

to

within

one

or

two

open

reading

frames

This

suggests

that

other

eight

regions

are

probably

quite

robust

when

designated

as

potential

lateral

transfer

regions

It

also

supports

the

usefulness

of

the

microarray

approach

All

regions

were

analyzed

using

Frame

Plot

-LRB-

Artemis

-RRB-

and

except

for

region

B

they

show

abnormalities

for

at

least

some

of

the

open

reading

frames

compared

to

the

G+C

bias

expected

for

the

and

codon

positions

of

Streptomyces

genes

Eight

regions

A

B

F

I

M

O

Q

and

T

contain

transposon

related

genes

near

to

or

within

the

region

Four

regions

H

N

P

and

R

are

flanked

by

highly

conserved

genes

such

as

a

ribosomal

protein

or

sigma

factor

genes

which

could

encourage

interspecific

recombination

Finally

five

regions

consist

largely

of

hypothetical

proteins

with

no

known

similarity

to

any

known

protein

as

yet

these

regions

are

G

J

L

S

and

W.

Region

L

is

particularly

interesting

as

there

is

a

central

core

of

conserved

gene

flanked

by

two

subregions

that

are

highly

not

conserved

One

of

these

genes

is

a

putative

spore

septum

determining

protein

while

the

rest

have

unknown

functions

Taken

as

a

whole

the

results

suggests

that

S.

coelicolor

may

have

recently

acquired

all

these

regions

either

by

transposition

or

by

interspecific/intraspecific

recombination

-LRB-

Wolf

et

al.

Zhang

et

al.

-RRB-

It

is

also

unlikely

that

they

were

acquired

from

any

of

the

four

species

studied

here

There

are

other

regions

that

could

potentially

be

identified

as

lateral

transfer

positions

using

less

stringent

criteria

and

a

wider

screening

of

genomes

might

help

to

support

these

additional

regions

as

being

involved

in

hotizontal

transfer

In

addition

such

a

wider

screen

might

allow

the

identification

of

possible

origins

of

these

regions

in

other

species.Table

of

the

Streptomyces

coelicolor

genome

identified

as

potentially

horizontally

transferred

regions

based

on

microarray

parallel

gene

absence

in

all

four

speciesRegionArea

of

chromosomeGenes

missinga

aSignificant

featuresRegion

insertion

sequenceRegion

ribosyl

transferaseRegion

excisionaseRegion

fstK-likeRegion

proteinsRegion

dnaZ

geneRegion

proteinsRegion

DNA

invertaseRegion

proteinsRegion

excisionaseRegion

ribosomal

proteins

operonRegion

excisionaseRegion

sigma

factor

whiGRegion

korSARegion

ribosomal

protein

bldBRegion

proteins

xylanaseRegion

proteinsaThis

is

calculated

from

the

available

normalized

gene

dataset

from

the

two

microarrays

Gene

conservation

in

the

terminal

regions

of

the

four

Streptomyces

species

As

has

been

mentioned

earlier

the

two

regions

at

either

terminus

are

much

less

well

conserved

than

the

central

core

region

these

extend

from

to

about

on

the

left

arm

of

the

chromosome

and

from

about

to

on

the

right

arm

The

boundaries

of

these

regions

are

not

absolutely

clear-cut

but

what

is

clear

is

that

as

one

moves

towards

the

centre

of

the

genome

gene

conservation

increases

beyond

these

points

This

can

be

clearly

seen

in

Fig.

where

the

gene

conservation

is

plotted

using

a

moving

window

for

the

four

species

but

it

is

also

clear

that

the

lack

of

conservation

is

not

uniform

across

the

terminal

regions

and

that

areas

of

higher

gene

conservation

can

be

identified

The

significant

interest

in

the

terminal

regions

arises

because

the

genomes

of

all

Streptomyces

that

have

been

examined

are

linear

and

the

problem

of

how

the

termini

of

such

a

molecule

replicate

is

of

particularly

importance

Recent

studies

have

indicated

that

two

genes

in

particular

tpgA

-LRB-

-RRB-

and

tapA

-LRB-

-RRB-

are

involved

in

this

process

-LRB-

Yang

et

al.

Bao

and

Cohen

-RRB-

tpgA

encoding

the

terminal

protein

that

covalently

binds

to

the

termini

of

many

linear

Streptomyces

replicons

is

conserved

across

all

four

species

In

S.

avermitilis

this

is

also

true

based

on

sequence

data

and

further

more

there

are

multiple

copies

of

tpgA

unlike

S.

coelicolor

The

signal

level

of

the

S.

avermitilis

gene

at

supports

the

presence

of

these

multiple

copies

The

signal

levels

for

the

other

three

species

are

between

about

and

which

supports

a

single

slightly

diverging

copy

of

this

gene

in

these

species

However

if

two

copies

are

present

then

the

sequence

divergence

may

be

higher

Furthermore

tapA

is

also

conserved

except

for

S.

maritimus

which

seems

to

be

more

divergent

at

It

should

be

noted

that

the

presence

of

these

two

genes

is

not

a

criteria

for

defining

a

genome

with

a

linear

topology

but

the

presence

of

one

or

both

is

certainly

suggestive

-LRB-

Dary

et

al.

Wang

et

al.

Huang

et

al.

Lin

and

Chen

-RRB-

Finally

ttrA

is

known

to

be

involved

in

chromosomal

transfer

and

is

found

very

close

to

the

telomere

of

S.

coelicolor

and

S.

avermitilis

This

is

also

conserved

in

all

four

species

suggesting

the

genetic

exchange

is

highly

important

in

Streptomyces

and

related

species

The

two

terminal

regions

encompass

the

major

areas

that

are

prone

to

deletion

in

many

Streptomyces

species

and

are

therefore

not

essential

except

for

linear

terminal

replication

and

genetic

exchange

Given

the

relatively

high

lack

of

conservation

of

genes

in

this

region

genes

that

are

present

in

all

four

species

represent

an

interesting

class

A

full

list

of

all

genes

conserved

in

all

four

species

in

the

terminal

regions

is

provided

in

Tables

and

There

are

hypothetical

genes

that

show

high

similarity

in

the

two

terminal

regions

Analysis

of

these

groups

of

conserved

genes

using

Artemis

-LRB-

The

Sanger

Institute

-RRB-

identifies

a

total

of

five

groups

of

genes

that

may

make

up

possible

single

transcriptional

units

These

are

and

In

addition

to

TpgA

and

TapA

it

is

possible

that

there

are

other

genes

involved

in

terminal

replication

and

these

may

be

among

the

conserved

genes

present

in

the

terminal

regions

Although

possible

candidates

can

be

deduced

from

a

direct

comparison

of

the

two

known

Streptomyces

genome

sequences

they

are

many

in

number

Using

the

microarray

analysis

of

the

Actinomycetes

in

this

study

the

candidates

can

be

reduced

significantly

From

candidates

in

Tables

and

two

possible

transcriptional

units

seem

to

be

potential

candidates

for

involvement

in

terminal

replication

these

are

-LRB-

hypothetical

proteins

-RRB-

and

-LRB-

including

hypothetical

proteins

an

AMP-binding

ligase

and

membrane

proteins

-RRB-

Gene

knockout

studies

may

be

able

to

identify

possible

functions

for

these

and

other

gene

candidates

especially

the

other

hypothetical

proteins

that

are

conserved

in

these

four

species.Table

from

the

-LRB-

a

-RRB-

left

terminal

-LRB-

b

-RRB-

right

terminal

region

of

Streptomyces

coelicolor

showing

microarray

conservation

in

all

four

species

-LRB-

a

-RRB-

putative

TetR-family

transcriptional

regulatory

hypothetical

hypothetical

hypothetical

putative

ABC

transporter

putative

integral

membrane

putative

penicillin-binding

hypothetical

putative

transmembrane

transport

hypothetical

putative

marR-family

transcriptional

hypothetical

hypothetical

putative

regulatory

polypeptide

araC

family

transcriptional

putative

TetR-family

transcriptional

putative

araC

family

transcriptional

putative

membrane

putative

iron-siderophore

permease

transmembrane

RNA

polymerase

principal

sigma

factor

hypothetical

putative

transmembrane

efflux

probable

sugar

transporter

sugar

binding

putative

membrane

hypothetical

secreted

putative

pyruvate

putative

lysR-family

transcriptional

putative

histidine

kinase

hypothetical

putative

response

putative

secreted

proline-rich

putative

polyprenyl

putative

RNA

polymerase

sigma

putative

hypothetical

putative

lysozyme

putative

integral

membrane

hypothetical

hypothetical

hypothetical

conserved

hypothetical

putative

membrane

hypothetical

hypothetical

putative

possible

hypothetical

hypothetical

hypothetical

hypothetical

hypothetical

putative

branched-chain

amino

acid

ABC

transport

putative

tetR-family

regulatory

putative

branched-chain

amino

acid

ABC

transport

putative

phosphotriesterase-family

putative

branched-chain

amino

acid

transport

ATP-binding

putative

DNA

repair

putative

branched-chain

amino

acid

transport

ATP-binding

hypothetical

secreted

putative

transcriptional

regulatory

conserved

hypothetical

putative

secreted

hypothetical

putative

metal

transporter

putative

hydrolase

-LRB-

b

-RRB-

putative

two-component

system

sensor

putative

secreted

solute-binding

putative

metal

transport

integral

membrane

putative

transport

system

integral

membrane

putative

ABC

transporter

ATP-binding

putative

AMP-binding

putative

non-ribosomal

peptide

conserved

hypothetical

conserved

hypothetical

putative

hypothetical

putative

ABC

transporter

ATP-binding

hypothetical

hypothetical

hypothetical

Tpg

proteinBold

indicates

groups

of

consecutive

genes

that

may

form

a

single

transcriptional

unit

Conservation

of

functional

groups

of

genes

across

the

four

Streptomyces

species

One

approach

to

analyzing

genetic

variation

across

these

four

Streptomyces

species

is

to

look

at

the

functional

groupings

of

genes

Such

an

approach

should

allow

the

identification

of

strain

versus

genus

specific

genes

especially

when

there

are

large

numbers

of

genes

with

related

functions

such

as

sigma

factors

or

where

there

are

two

copies

of

a

gene

such

as

ftsK

However

because

microarray

data

paints

a

broad

picture

across

a

whole

genome

it

is

essential

that

once

a

gene

or

genes

has

been

targeted

based

on

microarray

data

that

experimental

verification

by

other

means

is

carried

out

However

it

is

hoped

that

this

dataset

will

be

able

to

help

researchers

prioritize

their

gene

targets

better

The

genes

of

the

S.

coelicolor

chromosome

have

been

grouped

based

on

the

scheme

of

M.

Riley

and

colleagues

for

E.

coli

-LRB-

ecocyc.org

-RRB-

modified

for

S.

coelicolor

-LRB-

-RRB-

and

we

used

this

classification

The

genes

involved

in

ribosomal

proteins

synthesis

and

modification

should

be

highly

conserved

and

the

results

indicate

that

almost

all

of

them

are

present

in

all

four

species

-LRB-

Table

Supplementary

Fig.

-RRB-

The

only

exceptions

are

and

in

S.

avermitilis

and

and

in

K.

aureofaciens

Of

these

genes

and

represent

duplicate

genes

in

the

S.

coelicolor

genome

and

therefore

the

choice

of

the

microarray

sequence

will

have

had

a

significant

effect

on

the

heterologous

hybridization

There

is

no

obvious

explanation

for

the

failure

to

hybridize

of

the

other

two

genes

but

as

a

whole

this

dataset

supports

the

integrity

of

the

array

system

for

analysis

of

genome

content

as

these

genes

are

scattered

across

the

whole

Streptomyces

genome.Table

data

for

ribosomal

proteins

from

the

four

species

S.

avermitilisS

cattleyaS

maritimusK

probable

ribosomal

protein

putative

ribsomomal

protein

fragment

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

family

putative

ribosomal

protein

putative

ribosomal

protein

putative

ribosomal

protein

putative

ribosomal

protein

putative

ribosomal

protein

putative

ribosomal

protein

putative

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

putative

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

ribosomal

protein

Mean

hybridization

score

for

ribosomal

protein

Bold

values

indicate

that

the

signal

for

that

gene

is

more

than

below

the

mean

core

signal

for

that

species

and

such

a

value

is

suggestive

of

either

gene

absence

or

very

low

similarity

Table

shows

genes

identified

as

possible

sigma

factors

anti-sigma

factors

and

ant-sigma

factor

antagonists

The

genes

found

in

the

central

core

region

are

more

conserved

As

would

be

expected

the

major

sigma

factors

such

as

hrdA

hrdB

hrdC

and

hrdD

are

conserved

as

well

as

many

of

the

other

studied

sigma

factors

of

S.

coelicolor

such

as

are

sigA

sigE

sigF

sigG

sigR

sigT

and

whiG

Overall

fewer

regulation

genes

from

this

group

-LRB-

anti-sigma

factors

and

anti-anti-sigma

factors

-RRB-

are

conserved

than

sigma

factors

themselves

This

analysis

allows

the

identification

of

new

candidate

sigma

factors

for

further

study

outside

of

the

well

studied

ones

but

within

S.

coelicolor

and

in

other

species

Overall

the

results

support

the

hypothesis

that

there

is

a

core

of

sigma

factors

essential

to

keeping

protein

synthesis

in

Streptomyces

running

smoothly

The

functionality

of

the

rest

may

vary

and

include

complete

silence

of

some

gene

fragments

duplication

of

function

involvement

in

specific

secondary

metabolic

activities

and

species/genus

specific

functions.Table

across

the

four

species

of

genes

annotated

as

sigma

factors

or

related

proteins

in

Streptomyces

coelicolor

S.avermitilisS.cattleyaS

maritimusK

aureofaciens

putative

sigma

factor

putative

ECF

sigma

factor

putative

sigma

factor

putative

transcriptional

regulator

putative

RNA

polymerase

sigma

factor

putative

anti

anti

sigma

putative

regulator

of

putative

RNA

polymerase

sigma

factor

putative

anti-sigma

factor

antagonist

putative

anti

sigma

factor

antagonist

putative

RNA

polymerase

sigma

factor

probable

ECF-family

sigma

factor

probable

ECF-family

sigma

factor

putative

anti-sigma

factor

antagonist

RNA

polymerase

principal

sigma

factor

putative

RNA

polymerase

sigma

putative

ECF-sigma

factor

RNA

polymerase

ECF

sigma

factor

putative

RNA

polymerase

sigma

putative

RNA

polymerase

sigma

putative

RNA

polymerase

sigma

factor

RNA

polymerase

principal

sigma

putative

RNA

polymerase

sigma

putative

RNA

polymerase

sigma

putative

regulator

of

putative

anti

anti

sigma

factor

putative

RNA

polymerase

sigma

RNA

polymerase

principal

sigma

putative

RNA

polymerase

sigma

ECF

sigma

factor

putative

RNA

polymerase

sigma

factor

-LRB-

ECF

subfamily

-RRB-

putative

anti-sigma

factor

bldG

putative

anti-sigma

factor

antagonist

putative

RNA

polymerase

sigma

putative

anti-sigma

factor

putative

ECF

sigma

putative

ECF

sigma

putative

RNA

polymerase

ECF

sigma

factor

putative

RNA

polymerase

sigma

putative

anti

sigma

factor

antagonist

putative

RNA

polymerase

sigma

RNA

polymerase

sigma

factor

-LRB-

fragment

-RRB-

putative

ECF

subfamily

sigma

factor

putative

RNA

polymerase

sigma

factor

putative

anti

anti

sigma

factor

putative

sigma

factor

ECF

sigma

putative

ECF

sigma

putative

ECF

sigma

putative

ECF

sigma

factor

putative

ECF-sigma

possible

sigma

factor

putative

RNA

polymerase

ECF

sigma

factor

putative

ECF-subfamily

sigma

factor

anti-sigma

factor

anti-sigma

factor

putative

anti-sigma

factor

RNA

polymerase

sigma

factor

hrdB

major

vegetative

sigma

putative

sigma

putative

sigma

factor

putative

RNA

polymerase

sigma

putative

RNA

polymerase

sigma

factor

putative

RNA

polymerase

sigma

factor

putative

ECF-family

RNA

polymerase

sigma

factor

putative

ECF

sigma

factor

probable

RNA

polymerase

sigma

factor

anti-sigma

factor

anti-sigma

factor

putative

RNA

polymerase

secondary

sigma

factor

putative

anti-sigma

factor

antagonist

putative

anti

sigma

factor

antagonist

putative

anti-sigma

factor

antagonist

Mean

hybridization

score

for

ribosomal

protein

NABold

values

indicate

that

the

signal

for

that

gene

is

more

than

below

the

mean

core

signal

for

that

species

and

such

a

value

is

suggestive

of

either

gene

absence

or

very

low

similarity

A

conserved

gene

is

one

that

seems

to

be

present

in

all

four

species

Not

applicable

All

four

species

studied

here

undergo

differentiation

and

spore

formation

and

as

such

would

be

expected

to

retain

most

genes

involved

in

cell

division/sporulation/differentiation

This

is

supported

by

Table

K.

aureofaciens

shows

greater

gene

divergence

for

certain

genes

when

compared

to

the

three

Streptomyces

species

and

these

are

specifically

ftsI

-LRB-

-RRB-

and

a

putative

cell

division

protein

-LRB-

-RRB-

However

in

general

the

same

genes

in

all

four

species

show

a

higher

divergence

for

example

sapA

which

is

a

protein

associated

with

the

spore

surface

hydrophobicity

As

spore

morphology

varies

a

lot

in

the

Streptomyces

high

variability/gene

loss

in

such

a

gene

is

not

unexpected

Other

genes

that

show

higher

divergence

are

those

involved

in

partitioning

and

cell

division

This

suggests

that

the

genes

and

thus

the

proteins

involved

in

these

functions

may

differ

from

species

to

species

in

order

to

create

the

variation

seen

in

aerial

mycelium

and

spore

structure

across

Streptomyces

species

Specifically

an

ftsK

family

protein

gene

is

less

well

conserved

than

its

homologue

This

suggests

that

may

produce

the

major

ftsK

protein

Other

Fts

proteins

show

a

similar

pattern

with

at

least

one

homologue

being

well

conserved

This

may

well

help

an

understanding

of

the

relationships

between

the

genes

involved

in

cell

division

and

will

allow

better

identification

of

specific

targets

for

further

study

One

anomaly

that

stands

out

is

bldB

This

gene

consistently

shows

a

low

level

of

hybridization

A

comparison

of

the

bldB

gene

sequence

between

S.

coelicolor

and

S.

avermitilis

shows

a

nucleotide

identity

of

about

which

ought

to

give

a

signal

in

the

region

of

or

better

As

two

different

arrays

are

used

in

this

study

mechanical

problems

with

this

spot

can

probably

be

eliminated

as

the

source

of

the

anomaly

We

suggest

that

because

this

is

a

relatively

small

gene

the

PCR

product

chosen

for

both

arrays

may

be

the

reason

for

this

result

This

emphasizes

that

array

data

should

be

used

with

a

degree

of

caution

and

needs

to

be

backed

up

by

other

experimental

evidence

when

specific

genes

are

being

investigated.Table

across

the

four

species

of

genes

in

Streptomyces

coelicolor

annotated

as

involved

in

cell

division

sporulation

and

differentiation

S.

avermitilisS

cattleyaS

maritimus

K.

sapA

spore-associated

protein

precursor

putative

amino

bldD

putative

DNA

binding

putative

partitioning

or

sporulation

ftsZ

cell

division

ftsQ

sporulation

fts

W

putative

cell

division

ftsl

cell

division

Sfr

penicillin

binding

protein

mreD

rod

shape-determining

mreC

rod

shape-determining

protein

mreB

rod

shape-determining

putative

cell

division

trigger

putative

cell

division

ftsE

cell

division

ATP-binding

whiB

sporulation

regulatory

bldB

putative

RNA

polymerase

sigma

cell

division

protein

ftsH

bldG

putative

anti-sigma

factor

antagonist

putative

septum

site

determining

putative

morphological

differentiation-associated

putative

FtsW/RodA/SpoVE

family

cell

cycle

putative

partitioning

or

sporulation

putative

partitioning

or

sporulation

protein

ftsK/spoIIIE

family

protein

sporulation

associated

protein

mfC

aerial

mycelium

putative

cell

division-related

protein

putative

septum

determining

protein

putative

sporulation-related

protein

putative

sporulation-related

protein

putative

regulatory

bldM

putative

two-component

putative

septum

site-determining

protein

putative

septum

site-determining

BldKA

BldKC

bldKE

putative

peptide

transport

system

ATP-binding

protein

whiE

protein

VII

polyketide

cyclase

acyl

carrier

protein

polyketide

beta-ketoacyl

synthase

alpha

polyketide

ftsH

cell

division

protein

FtsH

homolog

whiG

RNA

polymerase

sigma

factor

bldB

putative

regulator

BldB

ftsK

whiH

sporulation

transcription

whiI

two-component

Mean

hybridization

score

for

ribosomal

protein

Bold

values

indicate

that

the

signal

for

that

gene

is

more

than

below

the

mean

core

signal

for

that

species

and

such

a

value

is

suggestive

of

either

gene

absence

or

very

low

similarity

The

genes

involved

in

DNA

replication

repair

restriction/modification

are

shown

in

Table

and

only

about

of

these

genes

are

not

conserved

relatively

well

across

all

four

species

This

is

to

be

expected

as

DNA

replication

and

repair

are

core

functions

Most

of

the

genes

that

show

higher

levels

of

gene

divergence

are

found

in

the

terminal

regions

of

the

linear

chromosome

and

probably

are

genes

that

perform

functions

that

are

not

essential

to

cell

survival

because

the

terminal

regions

of

Streptomyces

chromosomes

are

unstable

and

liable

to

deletion

without

lethality

Of

particular

interest

are

and

-LRB-

deoxiribopyrmidine

photolyases

-RRB-

these

repair

system

would

seem

to

be

absent

in

S.

lividans

and

S.

maritimus

but

a

homologue

is

present

in

S.

avermitilis

-LRB-

confirmed

by

the

genome

sequence

-RRB-

and

in

S.

cattleya

This

confirms

the

high

variability

found

for

this

repair

function

across

the

Streptomyces

-LRB-

Kobayashi

et

al.

-RRB-

A

similar

situation

of

high

variability

is

found

for

the

mutT

homologues

potential

hydroxy-dGTP

hydrolases

Knockout

of

this

gene

has

been

shown

to

increase

the

A

T

to

G

T

mutation

rate

and

thus

it

has

a

possible

repair

function

-LRB-

Kamiya

et

al.

-RRB-

The

genes

for

recA

-LRB-

-RRB-

recF

-LRB-

-RRB-

and

recR

-LRB-

-RRB-

are

present

in

all

four

species

however

the

recX

-LRB-

-RRB-

is

more

divergent

and

gives

a

low

signal

for

S.

cattleya

and

S.

maritimus

a

putative

DNA

recombinase

is

scored

as

absent

in

all

four

species

suggesting

that

there

is

redundancy

in

the

Streptomyces

genes

concerned

with

recombination

or

that

this

gene

is

transposon

related

The

latter

is

supported

by

low

homology

to

S.

avermitilis

putative

integrases/recombinases

There

are

four

genes

encoding

DNA

gyrases

on

the

microarray

namely

gyrA

DNA

gyrase

subunit

A

-LRB-

-RRB-

and

gyrB

DNA

gyrase

subunit

B

-LRB-

-RRB-

together

with

and

and

these

may

be

TopIV

homologues

involved

in

resolving

chromosome

concatenates

All

are

conserved

although

the

conservation

of

is

lower

Thus

both

sets

of

gyrase

genes

would

seem

to

be

important

As

expected

a

ruvB

Holliday

junction

protein

gene

and

a

ruvC

crossover

junction

endonuclease

are

conserved

across

all

the

species

Unexpectedly

although

probably

present

in

all

species

ruvA

is

much

more

divergent

that

the

other

two

gene

in

this

Holliday

junction

complex

This

diversity

is

unexpected

and

not

easily

explicable

except

by

the

fact

that

recombination

in

Streptomyces

may

occur

via

a

more

variable

mechanism

than

in

other

groups

of

bacteria

and

this

is

then

reflected

in

the

greater

divergence

of

ruvA

All

three

genes

annotated

as

a

DNA

polymerase

homologue

are

conserved

as

are

four

out

of

the

five

DNA

polymerase

III

homologues

suggesting

that

there

are

roles

for

all

of

these

conserved

genes

in

Streptomyces

Two

other

unclassified

DNA

polymerase

type

genes

and

are

also

conserved

and

thus

may

have

important

functions

There

is

however

more

diversity

among

the

helicases

and

methylases/methyltransferases

With

the

helicases

three

out

of

show

significant

divergence

and

therefore

most

of

the

helicases

probably

have

important

cellular

roles

Four

out

of

nine

methylases/methyltransferases

show

divergence

As

some

of

these

genes

may

be

involved

in

the

DNA

modification

part

of

restriction/modification

such

diversity

across

strains

in

not

unexpected

Finally

four

out

of

six

ligases

show

divergence

perhaps

reflecting

the

fact

that

the

origin

of

a

number

of

these

ligases

might

be

from

bacteriophages.Table

across

the

four

species

of

genes

in

Streptomyces

coelicolor

annotated

as

involved

in

DNA

replication

repair

restriction

and

modification

S.

avermitilisS

cattleyaS

maritimusK

putative

deoxyribodipyrimidine

photolyase

putative

methyltransferase

putative

deoxyribodipyrimidine

putative

excinuclease

ABC

subunit

A

putative

formamidopyrimidine-DNA

glycosylase

putative

DNA

repair

putative

DNA

protection

protein

uracil

DNA

glycosylase

putative

helicase

-LRB-

fragment

-RRB-

putative

DNA

polymerase

III

beta

chain

putative

DNA

putative

MutT-like

protein

G/U

mismatch-specific

DNA

uracil-DNA

putative

DNA

damage

inducible

mutT-like

putative

primosomal

ruvB

holliday

junction

DNA

ruvA

holliday

junction

DNA

helicase

ruvC

crossover

junction

putative

DNA

polymerase

putative

DNA

polymerase

putative

DNA

repair

protein

putative

DNA

glycosylase

putative

DNA

polymerase

ABC

excision

nuclease

subunit

putative

DNA-methyltransferase

DNA

polymerase

DNA

putative

DNA

repair

hydrolase

-LRB-

fragment

-RRB-

putative

helicase

putative

helicase

putative

transcriptional-repair

coupling

factor

putative

DNA

repair

protein

putative

DNA-binding

putative

DNA

polymerase

putative

DNA

putative

DNA

probable

DNA

topoisomerase

putative

putative

recomination

DNA

gyrase

subunit

DNA

gyrase

subunit

DNA

polymerase

chromosomal

replication

initiator

protein

-LRB-

fragment

-RRB-

ATP-dependent

helicase

putative

mutT-like

putative

mutT-like

protein

putative

DNA

invertase

putative

DNA

polymerase

related

protein

putative

putative

ATP-dependent

DNA

helicase

putative

bifunctional

protein

glycosylase

I

putative

ATP-dependent

DNA

putative

ATP-dependent

DNA

putative

ATP-dependent

DNA

putative

DNA

methylase

putative

DNA

putative

ATP-dependent

DNA

putative

formamidopyrimidine-DNA

DNA

RecX

protein

putative

ATP-dependent

SOS

regulatory

protein

ribonucleotide

probable

ATP-dependent

DNA

helicase

probable

DNA

DNA

gyrase-like

putative

DNA

polymerase

putative

methylated-DNA-protein-cysteine

methyltransferase

putative

helicase

forward

MW

putative

DNA

recombinase

putative

methylated-DNA-protein-cysteine

methyltransferase

putative

ATP-dependent

helicase

putative

DNA

ligase

putative

DNA

methylase

putative

DNA

ligase

probable

ATP-dependent

DNA

putative

DNA

ligase

Mean

hybridization

Bold

values

indicate

that

the

signal

for

that

gene

is

more

than

below

the

mean

core

signal

for

that

species

and

such

a

value

is

suggestive

of

either

gene

absence

or

very

low

similarity

Table

shows

the

genes

involved

in

peptidoglycan

and

teichoic

acid

synthesis

In

this

area

of

metabolism

there

is

also

a

relatively

high

level

of

conservation

of

genes

particularly

the

murA

murB

murD

murE

murF

murG

and

murX

genes

Also

conserved

are

the

shape-determining

genes

and

which

may

form

an

operon

This

probably

represent

a

core

of

genes

together

with

the

genes

involved

in

biosynthesis

of

the

cell

wall

that

are

needed

to

give

a

basic

structure

to

the

cells

of

any

Streptomyces

species

The

penicillin

binding

proteins

show

a

higher

degree

of

variability

except

for

and

The

peptidases

and

and

the

D-alanine

:D

lactate

ligase

all

show

a

low

level

of

gene

conservation

perhaps

because

they

are

involved

in

relatively

broad

cellular

functions

and

not

under

a

great

deal

of

selective

pressure.Table

across

the

four

species

of

genes

in

Streptomyces

coelicolor

annotated

as

involved

in

peptidoglycan

biosynthesis

S.

avermitilisS.cattleyaS

maritimusK

putative

oxidoreductase

putative

peptidoglycan

binding

protein

putative

penicillin-binding

putative

oligosaccharide

deacetylase

putative

putative

secreted

penicillin

binding

protein

putative

cell

division

putative

peptidodoglycan-binding

membrane

protein

putative

rod

shape-determining

putative

glycosyl

putative

penicillin

binding

protein

rod

shape-determining

rod

shape-determining

protein

rod

shape-determining

putative

putative

probable

penicillin-binding

putative

transpeptidase

putative

D-alanine

:D

lactate

ligase

putative

D-alanine

:D

alanine

dipeptidase

putative

D-alanyl-D-alanine

carboxypeptidase

putative

penicillin-binding

putative

penicillin-binding

protein

putative

penicillin

binding

protein

putative

secreted

putative

D-alanyl-D-alanine

carboxypeptidase

murB

putative

penicillin-binding

putative

penicillin-binding

protein

putative

muramoyl-pentapeptide

carboxypeptidase

D-alanine-D-alanine

putative

UDP-N-acetylmuramoyl-L-alanine

putative

D-alanyl-D-alanine

Mean

hybridization

Bold

values

indicate

that

the

signal

for

that

gene

is

more

than

below

the

mean

core

signal

for

that

species

and

such

a

value

is

suggestive

of

either

gene

absence

or

very

low

similarity

Conserved

genes

with

no

known

function

Genes

with

no

known

function

and

no

homologue

outside

of

S.

avermitilis

that

are

conserved

across

the

other

three

Streptomyces

species

should

represent

genes

important

to

specifically

being

a

myceliate

Actinobacteria

and

the

phenotype

of

gene

knockout

strains

for

these

genes

will

be

particularly

interesting

in

terms

of

Streptomyces

biology

Based

on

the

dataset

here

genes

can

be

identified

as

annotated

as

either

conserved

hypothetical

genes

or

non-conserved

hypothetical

genes

and

these

are

shown

in

Supplementary

Table

The

proportion

of

these

genes

that

are

conserved

across

all

four

species

are

and

for

the

left

terminal

region

left

Streptomyces

specific

region

core

region

right

Streptomyces

specific

region

and

right

terminal

region

respectively

There

is

also

a

low

frequency

of

conserved

hypothetical

genes

in

the

left

terminal

region

and

right

Streptomyces

specific

region

and

respectively

compared

to

for

the

left

Streptomyces

specific

region

for

the

core

region

and

for

the

right

terminal

region

It

is

clear

that

there

is

a

need

to

further

screen

these

genes

by

increasing

the

range

of

Streptomyces

species

analyzed

by

microarray

hybridization

This

will

reduce

the

number

to

a

manageable

number

and

will

allow

prioritization

of

genes

for

knockout

and

detailed

phenotypic

analysis

Another

approach

to

the

problem

of

identifying

functionally

important

genes

is

by

the

pinpointing

of

functional

groups

of

such

genes

that

may

form

a

transcriptional

unit

Blocks

of

three

or

more

hypothetical

genes

that

are

conserved

across

all

species

were

identified

and

are

shown

in

Table

It

is

possible

that

these

groups

represent

conserved

functional

groups

of

genes

essential

to

core

functions

that

make

Streptomyces

different

from

other

bacteria

They

are

found

mostly

in

the

area

between

the

Streptomyces

terminal

regions

and

the

central

core

region

There

are

seven

groups

of

conserved

hypothetical

genes

larger

than

five

genes

-LRB-

and

-RRB-

It

is

likely

due

to

the

proximity

of

various

genes

around

that

this

complex

is

involved

in

cell

division

development

and

DNA

partitioning

The

function

of

the

others

groups

is

unknown

Interestingly

of

these

gene

groups

are

upregulated

shifting

from

exponential

phase

to

stationary

phase

or

under

stress

shift

as

indicated

by

Karoonuthaisiri

et

al.

-LRB-

-RRB-

Table

genes

in

S.

coelicolor

conserved

as

a

group

in

the

four

species

analyzedGenes

-LRB-

SCO

-RRB-

Operon

structureaLinked

function

if

operon

operonBoth

flanks

of

rRNA

gene

operon

operonGlucose

operonClose

to

heme

operonPenicillin

binding

operonHomology

to

Mycobacterium

operonDNA

operonPossible

DNA

binding

operon

a

Gene

structure

from

Artemis

is

compatible

with

an

operon

type

structure

with

possible

appropriate

ribosome

binding

sitesb

Inside

or

linked

to

the

conserved

genes

is

a

gene

-LRB-

s

-RRB-

of

known

function

Conservation

of

genes

involved

in

secondary

metabolism

and

similar

functions

Genes

that

are

involved

in

secondary

metabolism

and

antibiotic

production

are

widely

distributed

in

the

Streptomyces

and

many

if

not

most

may

have

been

involved

in

horizontal

transfer

However

there

is

significant

similarity

between

genes

involved

in

similar

pathways

and

thus

significant

cross-hybridization

may

occur

between

similar

metabolic

pathways

A

large

number

of

genes

are

also

involved

in

secondary

metabolism

-LRB-

-RRB-

and

polyketide

synthesis

-LRB-

-RRB-

in

the

S.

coelicolor

genome

These

are

grouped

together

in

pathway

groups

and

are

displayed

in

Supplementary

Fig.

Genes

identified

as

secondary

metabolic

genes

but

existing

on

their

own

and

not

in

a

group

of

secondary

metabolic

genes

have

been

eliminated

to

simplify

the

analysis

leaving

only

genes

involved

in

these

functions

with

two

or

more

genes

together

in

a

group

These

include

specific

pathways

producing

secondary

metabolic

products

such

as

melanin

actinorhodin

CDA

and

Red

pathway

Many

of

the

other

potential

pathways

have

not

been

studied

in

detail

and

the

functions

of

these

genes

are

unknown

Because

of

evolutionary

similarity

the

presence

of

genes

hybridizing

to

a

particular

pathway

does

not

mean

that

the

specific

pathway

is

present

but

possibly

that

a

related

one

is

may

be

present

Similarly

a

high

level

of

hybridization

can

mean

either

a

very

close

relationship

between

the

pathways

in

the

two

species

or

the

presence

of

multiple

copies

of

related

pathways

In

general

terms

S.

maritimus

shows

the

greatest

absence

of

secondary

metabolic

pathways

that

are

present

in

S.

coelicolor

Interesting

S.

cattleya

and

K.

aureofaciens

seems

to

have

pathways

related

to

many

of

the

S.

coelicolor

secondary

metabolic

pathways

present

in

their

genomes

although

they

are

phylogenetically

more

distant

than

S.

maritimus

The

actinorhodin

pathway

would

seem

to

be

absent

from

S.

avermitilis

-LRB-

as

expected

from

the

genome

data

-RRB-

S.

cattleya

and

S.

maritimus

although

some

related

genes

do

seem

to

be

present

in

K.

aureofaciens

The

WhiE

pathway

is

conserved

in

all

species

but

with

some

genes

showing

a

very

low

level

of

hybridization

in

certain

cases

and

these

include

whiE

protein

VII

and

the

acyl

carrier

protein

Genes

from

the

Red

pathway

show

varying

levels

of

hybridization

suggesting

that

distantly

related

pathways

may

be

present

in

these

species

The

CDA

pathway

is

conserved

in

all

four

species

and

in

certain

cases

the

genes

seem

to

be

over

represented

suggesting

multiple

examples

of

the

same

type

of

pathway

in

S.

cattleya

and

S.

maritimus

The

presence

of

similar

pathways

at

a

level

of

about

for

K.

aureofaciens

supports

the

well

established

idea

that

horizontal

gene

transfer

of

secondary

metabolic

pathways

may

have

played

a

significant

role

in

the

evolution

of

the

Streptomyces

and

any

related

genus

Because

the

natural

environment

of

Streptomyces

is

the

soil

they

are

thought

to

play

an

important

role

in

the

recycling

of

lignocellulose

material

However

there

is

relatively

little

information

on

what

genes

are

involved

in

this

process

Interestingly

and

which

encode

tyrosinase

-LRB-

monophenol

monooxygenase

-RRB-

and

its

cofactor

-LRB-

-RRB-

-LRB-

Leu

et

al.

-RRB-

are

conserved

across

the

three

Streptomyces

species

and

probably

also

Kitasatospora

-LRB-

-RRB-

On

the

other

hand

the

duplicate

-LRB-

-RRB-

and

-LRB-

-RRB-

genes

found

in

S.

coelicolor

are

not

conserved

and

are

phylogenetically

distinct

from

and

found

in

other

Streptomyces

-LRB-

unpublished

results

-RRB-

This

perhaps

represents

a

divergence

of

function

between

this

two

gene

pairs

S.

coelicolor

does

not

produce

a

detectable

amount

of

black

melanin

pigment

and

these

results

suggest

that

these

enzymes

may

be

involved

in

the

metabolic

conversion

of

lignocellulose

byproducts

rather

than

pigment

formation

Evolutionary

conservation

of

these

genes

to

serve

this

function

under

particular

conditions

of

induction

would

make

more

sense

than

retention

of

inducible

black

pigment

formation

Other

enzymes

with

a

possible

role

in

the

lignocellulose

cycle

that

are

conserved

across

the

species

are

shown

in

Table

These

include

many

oxygenases

that

may

have

a

role

in

producing

oxygen

radicals

capable

of

attacking

lignin

genes

involved

in

the

sensing

and

breaking

down

hydrogen

peroxide

cellulose

metabolism

genes

cellobiose

metabolism

genes

etc.

Those

found

in

the

terminal

regions

may

represent

gene

groups

that

are

not

conserved

in

a

syntenous

manner

and

subject

to

horizontal

gene

transfer

while

those

within

the

core

and

intermediate

regions

may

be

part

of

the

basic

group

of

genes

essential

to

Streptomyces

in

the

soil

environment

Lignocellulose

degradation

is

a

difficult

topic

to

study

in

the

Actinomycetales

and

therefore

these

candidate

genes

may

help

to

solve

some

of

the

problems

associated

with

this.Table

conserved

in

the

four

Streptomyces

species

that

are

potentially

involved

in

lignocellulose

binding

-LRB-

monophenol

monooxygenase

-RRB-

peroxide

sensing

that

the

oxygeneases

included

as

possible

enzymes

that

make

be

able

to

attack

lignin

are

all

unclassified

yet

as

to

their

real

function

The

core

region

is

in

bold

Conclusions

This

study

confirms

that

within

the

Streptomyces

analyzed

here

there

is

conservation

of

a

core

set

of

genes

in

the

middle

of

the

linear

S.

coelicolor/S

avermitilis

chromosome

structure

This

is

associated

with

a

much

higher

diversity

of

gene

in

the

terminal

regions

of

the

linear

chromosome

Linking

these

regions

are

two

intermediate

regions

where

there

seems

to

be

conservation

of

genus

specific

genes

and

gene

clusters

This

study

also

identifies

candidate

genes

that

may

be

possibly

involved

in

terminal

replication

and

other

myceliate

growth

related

functions

based

on

a

classification

of

genes

into

conserved

and

conserved

groups

This

study

also

provides

insights

into

which

genes

in

Streptomyces

play

a

more

significant

role

in

the

biochemical

network

of

S.

coelicolor

Streptomyces

and

myceliate

Actinobacteria

in

general

Finally

the

degree

of

gene

conserved

detected

between

the

four

species

implies

that

that

genome

model

of

S.

coelicolor

may

extent

well

beyond

the

borders

of

the

Streptomyces

It

includes

at

least

one

Kitasatospora

species

furthermore

a

similar

structure

by

microarray

analysis

has

been

found

for

Saccharomonospora

viridis

and

Streptosporangium

roseum

but

not

Streptomyces

rimosus

-LRB-

unpublished

data

-RRB-

Thus

the

microarray

approach

to

genome

content

analysis

and

exploration

of

genome

evolution

may

be

fairly

widely

applicable

in

the

various

Actinomycete

genus

close

to

Streptomyces

that

undergo

complex

morphogenesis

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

ESM

-LRB-

PDF

kb

-RRB-