Specificity of DNA uptake in genetic transformation of gonococci

Vol. 86, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 15, 1979

Pages 97-104

SPECIFICITY OF DNA UPTAKE IN GENETIC TRANSFORMATION OF GONOCOCCI Thomas J. Dougherty,

Anneleen

Asmus and Alexander

Tomasz

The Rockefeller University New York, New York 10021

Received

November 20,1978

Summary. Genetic transformation of gonococci to streptomycin resistance was inhibited by homo logous DNA or by DNA from related Neisseriae, but not by high concentrations of heterologous DNAs. Gonococci were capable of adsorbing large quantities (up to about 50 ng per 108 cells) of both homologous and heterologous DNA, which could not be eluted by strong shearing forces. Treatment with externally added DNase removed virtually all the heterologous DNA while a small fraction of the homologous DNA, not influenced by the presence of excess heterologous DNA, remained cell-bound in a form resistant to nuclease treatment. Competing homologous DNA suppressed nuclease-resistant binding. These findings suggest that gonococci have two types of DNA binding components at their surface. Competence of gonococci for genetic transformation undergoes a rapid decay if the cells are incubated with homologous (but DNA. -not with heterologous) INTRODUCTION.

Genetic

and adsorption

of exogenous

surface. this

Recent

step.

stranded

ribonucleotides

or double

duplexes

compete

of specificity uptake

tivity

of exogenous

the

a surface

located

binding

sites

of interaction

between

have two kinds

of DNA binding

components degree

(7).

A recent,

evidence

protein

exogenous

at the cell

of specificity

the DNA binding

(4,12).

stranded

system

degree

in which

DNA or DNA isolated

elegant

investigation

that

We describe

DNA and competent

poly-

An even higher

base sequence (3).

seem to

hetero

transformation

indicating

in

sites

double

to homologous

of a specific

bacterial

by the capture

polyribo-polydeoxyribonucleotide

in the Hemophylus

produced

initiated

polydeoxynucleotides,

these

species

recognition

a high

pneumococci

DNA was restricted

related

and Deich,has is

for

is

to binding

revealed

stranded

was observed

taxonomically Sisco

have

In competent

to double

do not

of bacteria

DNA molecules

experiments

adsorption

be restricted

transformation

the basis in

by H. Smith, of this

the exogenous

here gonococci

from

still which

selecDNA by

another appear

type to

components. 0006-291X/79/010097-08$01.00/0 97

Copyright All rights

@ 1979 by Academic Press, Inc. in any form reserved.

of reproduction

Vol.

86,

No.

1, 1979

BIOCHEMICAL

AND

Materials 51 colony University

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

and Methods. 1) Bacterial strains: Neisseria gonorrhoeae strain type T2 and T4 were provided by Dr. Richard B. Roberts of Cornell Medical College. A streptomycin resistant (STRR) strain (M.I.C. > 1 mg/ml) of this organism was constructed by transformation. N. gonorrhoeae MHD 316 (Hypoxanthine', Proline', Uracil-, Arginine') was obtained from Dr. Wesley Catlin, Medical College of Wisconsin, Milwaukee, Wisconsin. N. meningitidis was obtained from Dr. Emil Gotschlich of this university. NT subflava (ATCC 10555) was from the American Type Culture Collection. Bacteria were maintained by daily transfer on solid medium in candle jars at 37'C. 2) Medium The liquid medium used in this study was Brain Heart Infusion (DIFCO) with 1% defined supplements (Isovitalex) and 4 mM MgC12. For agar plates, GCBA Agar (Baltimore Biological Laboratories) with defined supplements was used. 3) Transformation Procedure The procedure for obtaining competent cells was essentially that used by Sarubbi Clonal types T2 were --et al. (6). scraped from an 18 hr GCBA plate and suspended in prewarmed BHI-supplements-Mg*. The cells were routinely suspended to a Nephelos value between 100 and 200 using a Coleman Nepho-Colorimeter. A value of N = 100 represents approximately 1.5 x 108 colony forming units. One milliliter of cells was added to tubes containing the DNA and incubated at 37°C for 20 minutes. The reaction was terminated with 50 ug/ml pancreatic DNase (Worthington 2X crystallized) and incubated 5 mins at 37'C. The cells were then diluted ten and one hundredfold in Brain Heart Infusion and 0.1 ml of each dilution added to 4 ml of soft GCBA agar at 48"C, mixed and immediately layered onto the surface of plates containing 20 ml of GCBA agar plus supplement. The plates were incubated at 37'C for at least 7 hours to allow phenotypic expression and then each plate received 5 mls of soft GCBA agar containing sufficient streptomycin to give a final concentration of 300 pg/ml throughout the plate. After 4 days of incubation, colonies were counted with a New Brunswick colony counter. 4) DNA Binding and U take Assay Cells from agar piates were suspended --%-(cell conztration: 3 X 10 viable units per ml) in Beef Heart Infusion with supplements and 4 mM MgC12. Radioactively'labeled DNA was added to 2 mls of this suspension and incubated for 20 minutes. For total binding, the cells were chilled and washed by centrifugation (12,000 X G, 10 mins, 4°C) 3 times with ice cold medium. The pellet was resuspended in cold saline and an aliquot transferred to tubes containing ice cold 10% CC13COOH (2 mls). After 10 mins on ice, the precipitate was collected on Whatman GFfA glass fiber filters, washed 2X with cold 10% CC13COOH and 2X with 95% ethanol. Radioactivity was assayed by placing the discs in 10 mls of toluene based scintillation fluid For DNase reusing a Nuclear-Chicago Mark II scintillation spectrometer. sistant uptake, the cells were incubated with labeled DNA for 20 mins and then The cells were then chilled and received 50 )Ig/ml DNase for 5 mins at 37°C. vrocessed as described above for total bindinn. 5) DNA preparations. E. coli B, g. p erfringens, & luteus DNAs were .' obtained from Sigma Chemical Co. h viral DNA was from Miles Laboratories. All were dissolved in saline. DNA was extracted from Neisseria species and Str. pneumoniae by sodium dodecyl sulfate-deoxycholate lysis of the cells (5). The DNA was suspended in 150 mM NaCl-15 mM sodium citrate and dialyzed at 4OC against 2 changes of the same buffer (500 volumes each) and 2 changes of saline 260 nm in a Zeiss PMQ-2 spectrophotometer where one absorbance unit = 50 & DNA. Radioactive DNA was obtained by biosynthetic labeling (8). 3H-pneumococcal DNA was from Str. pneumonia1 TVS (thymidine requirer) labeled with 3H- thymfdine (56 Ci/m mol= England Nuclear) in CpH8. Specific activity of the DNA reparation was 2.6 X 106 CPM/ug. N. gonorrhoeae MHD 316 was labeled with 3 H uracil (23.3 Ci/m mol- New Englazd Nuclear) in Beef Heart Infusion plus supplements. Specific activity of this DNA preparation was 1.43 X lo5 CPMfug. For testing the shear-sensitivity of DNA adsorbed to the cell surface, the cells were vortexed on a Vortex Jr. Mixer (maximum speed setting) for 5 seconds after each resuspension in the washing procedure. Control cells were gently

98

BIOCHEMICAL

Vol. 86, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE EFFECT

source Transforming

N.

OF HETEROLOCOUS DNA PREPARATIONS ON THE GENETIC TP.ANSFORMATION OF CONOCOCCI

Source Competing

of DNA

Number of (Concentration 0

---

---

---

3.01

3.11

2.30

1.76

---

4.3

3.22

2.32

1.90

PI. luteus

---

2.90

2.50

2.60

2.40

Str.

---

3.26

3.0

2.30

2.30

A

---

2.18

1.81

1.99

1.68

gonorrheae

---

0.8

0.5

0.13

0.08

coli

B

perfringens

pneumoniae

Phage N. Homologous,

STRR DNA (with

1 ~g/ml

and

Transformants (X 105) per ml of competing DNA pg per ml) 2 50 -10 -100 ---

*One

Cl.

of

of DNA

1.94

gonorrhoeaea E.

a

1

the

and without

DNas)

assays

performed

resuspended by finger agitation. This DNA in the pneumococcal transformation

vortexing system.

Effect

Results.

of gonococci. gonococcal

transformation

competing

of heterologous Competent

DNA (carrying a concentration

sufficient

see Fig.

2) and various

heterologous

tion

of these

was tested.

preparations

competing

Table

when used at loo-fold number

of transformants

DNA; the reason In contrast, from

excess

two Neisseria

for

this

Figure

effect

is

on the

assay, and

of transforma-

heterologous

DNA frequency

increase of competing

at the present

homologous

to gonococci

99

DNA concentration:

transformation

not understood

related

transformation

of transforming

the frequency

A substantial

that

Methods.

concentrations

at low concentrations

1 demonstrates

taxonomically

marker;

none of the six

concentration,

under

the transformation

DNAs to suppress

was noted

to mixtures

resistance

effect

a concentration

remove surface-bound

DNAs at different

inhibitory

at

DNA on the genetic

to saturate

1 shows that

had marked

would

exposed

the streptomycin

1 pg/ml,

the ability

were

added

as described

and homologous

gonococci

was

---

in the heterologous time.

DNA and DNA isolated significantly

even

inhibited

Vol.

86,

No.

1, 1979

BIOCHEMICAL

KG Figure

Competition

1.

DNA was purified gonococci . 1 A. 1 ug StrR DNA B. 1 ug StrR DNA C. 1 pg StrR DNA.

S trs

Cells 37Oc.

were

assayed

Reaction

gonococcal

Specificity can adsorb only ternally

added

competing, (both

from gonococci) cells

that

related

per

COMMUNICATIONS

ml

Neisseria

species.

Gonococcal

DNA and 1, 5 and 10 Dgs of StrS

g.

Gonococcal

DNA and 1, 5 and

E. meningitidis

10 ugs of StrS

after 20 minutes for StrR transformants was terminated with 50 ug/ml DNase.

and the degree

of the competing of DNA uptake

pancreatic

had DNA bound

2 demonstrates

of both

bound DNase,

inhibition were

homologous

in a form p resumably

DNA suppresses

and heterologous), causes

of inhibition

subflava

of incubation

at

was proportional

to

DNAs.

Table

quantities

DNA becomes

non-radioactive

homologous

DNA

RESEARCH

from 5. subflava and N. meningitidis, as well as from ml of T2 Gonococci (N = iO5) were added to tubes containing: Gonococcal DNA and 1, 5 and 10 ugs of StrS gonococcal

substantial

homologous

BIOPHYSICAL

COMPETING

of DNA from

transformation

the concentration

AND

only

resistant

the amounts homologous

to shearing

100

competent

gonococci

and heterologous to treatment

due to cellular

of nuclease-resistant subjected

that

DNA but with

uptake.

ex-

While

of surface-adsorbed competing

DNA (i.e.

DNA binding. forces,

DNA

there

DNA When

was no

BIOCHEMICAL

Vol. 86, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2 __BINDING MD uww Radioactive (uglml)

DNA

OF DNA bY COMPETENT GONOCOCCJ

Competing DNA (w/ml)

-_--_none

pneumococcal (1.0 ug)

45

0.16*

gonococcal (10)

26.5

0.32

pneumococcal (10)

22.3

0.20

none

65.7

4.1

gonococcal (5 )

34.1

2.1

pneumococcal (5 )

28.1

3.8

E. coli (5 )

21.6

4.3

gonococcal (In, up)

*less

than 1% DNase resistant

substantial

loss

of counts

CPM vortexed

sample;

tained

T4 colony

using

Saturation

binding

CPM control

sample).

20568 type

large

sites

Figure

radioactive

DNA).

At cell

concentrations

saturation

of this

binding

capacity

amounts

concentration,

control

The same results

(19852

were

ob-

2 shows that

gonococci

of DNA (as measured

by the adsorption

of 3 X lo8 viable

occured

at about

a maximum level

are capable

bacteria

50 pg/ml

of genetic

of

per ml,

pneumococcal

transformation

DNA. was

by 1 ng DNA per ml.

Decay of gonococcal

homologous

resuspended

cells.

relatively

Portions

background.

to a gently

of binding

attained

represents

as compared

of DNA binding

At the same cell

DNA Uptake (nuclease resistant binding, ng per 2 x 108 cells)

DNA Binding (Cell-associated DNA, ng per 2 x 108 cells)

competence

of a gonococcal

culture

STRR DNA was added

was determined

(Fig.

3).

to undergo were

preincubated

at intervals

A decline

genetic with

and the number

in STRR transformants

101

transformation homologous

STRS DNA,

of STRR transformants occurred

with

in-

Vol. 86, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1055 Km

104 -i f P

----mm_ ---me .----

---___...._

. . . . . .I I----

\ 5\

*-a

E a103I 8 5-

‘l

2 f E 1025-

I 0.01 I

0.05

0.1

0.5

1

2

5

10

2030 PREINCUBATION

03 Figure

2.

Saturation

of Binding

and Transformation

Total binding of increasing DNA (2.26 X lo6 CPM/Hg) to 2 mls described under Methods (e--a). of StrR gonococcal DNA were added of transformants to streptomycin cubations were for 20 minutes at Figure

3.

Decay of Competence

DNA

WITH

(minutes)

in Competant

Gonococci

concentrations of radioactive pneumococcal of T2 Gonococci (N = 180) was measured as For transformation, increasing concentrations to (N = 165) StrS T2 Gonococci and the number resistance was assayed (M). All in37OC.

by Pretreatment

with

Homologous

DNA.

Cultures of StrS Gonococci (N = 150) received 1 pg homologous StrS DNA at time 0. At intervals, 1 Dg of homologous StrR DNA was added and the number of transformants after 25 minutes of incubation at 37'C was assayed (v). For comparison purposes, 1 pg of pneumococcal DNA was added to 1 ml of competent cells, and 1 Hg of StrR gonococcal DNA added at indicated intervals and assayed for transformants @--+). All incubations were terminated with 50 rig/ml DNase.

creasing

time

of preincubation

STRS DNA did

not

Discussion.

The observations

petent These

gonococci two receptors

heterologous

cause

contains

in STRS DNA.

a similar

loss

DNA adsorb

here

2 independent

to receptor

with

the heterologous

of competence.

reported

have several

Preincubation

kinds

contrasting 1, while

102

suggest

that

the surface

of DNA binding features. binding

I)

sites Both

to receptor

of comor receptors.

homologous

and

2 seems to be

BIOCHEMICAL

Vol. 86, No. 1, 1979

restricted

to homologous

ii)

Large

not

all,

quantities of these

a portion

cells

of DNA can adsorb molecules

or about

concentration)

were

from pneumococcal some fashion. bound

iii)

Deterologous

formation.

In contrast,

Maximum amounts while

homologous decline

(but

high

in levels

capable pendages studies

It

(2)

with

for

trans-

receptor

2.

at physiological

temperatures

inhibited

at 0“C (unpublished

the specificity

of receptor (Fig.

DNA.

2 for

3) that

rapid

by incubation

The mechanism

of this

with phenomenon

time.

has been several

was found (10)

and it

of Tl and T2 cells dealt

of genetic

1 even at low temperature

may be initiated

have revealed

bacterium.

cells.

1 has virtually

competes

by the finding

of gonococci

studies

of DNA binding

have

for

heterologous)

of transformability

(pili)

are

transformability

transformation

this

only

in

out by the finding

to receptor

to receptors

illustrated

at the present

and further

uptake

is

is ruled

DNA molecules

evidence

remove DNA

protected

DNA or on the frequency

and transformation

-not with

understood

Genetic (10)

homologous

DNA molecules of gonococcal

is not

of homologous

that

of

the surface

T2 and T4 (non-piliated)

the medium or attached

v) A further

homologous

in both

of DNA from

the DNA is

DNA

because

(vortexing)

that

DNA per

at saturating

presumably

protection

2 seems to function

DNA uptake

observations).

forces

in shear

of DNA can bind

receptor

cells

if

DNase, while

to 4 ng gonococcal

to the

indicates

resistant

DNA in

on the uptake

This

of pili

shear

no effect

both

(9).

species.

1 but most,

to external

to be no loss

to shearing

related

receptor

accessible

2 (amounting

appeared

subjected cells

DNA is

at a site

via

to DNase treatment,

There

The role

that

since

resistant

taxonomically

to gonococci

to receptor

transport.

when the cells

(O°C),

remain

from

6% of the DNA adsorbed

becomes

intracellular

iv)

DNA or DNA isolated

of DNA attached

2 X lo8

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

physiological

only all

colonial

the four

has been suggested may play

a role

parameters

103

described

interesting

that

while

first

that

by Sparling

features types colonial

of DNA 1 and 2 have types

the surface

in DNA uptake of gonococcal

(1).

were apSeveral

transformation

(11).

Vol. 86, No. 1, 1979

The novelty specificity

BIOCHEMICAL

of the observations

of gonococcal

report,

gonococcal

genetic

transformation

description iates

considered

transformation

(3).

the latter

is

in Hemophylus

either

the

case

(3, 9 ).

Acknowledgements.

Public

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Institutes Health

Literature

This

Service

Award

homologous

of H. Smith

elements

(#AI

system,

in Sexually

T.J.D.

Transmitted

Scocca

of

detailed

and his

basis

colleagues

assoc-

is

of selective indicate

in this

report

sources

via

in their

has been supported 12932).

the mechanism

molecular

described

growing

In this

molecules.

enzyme or a base-sequence

from heterologous

in gonococci

the apparent

In the first

and his

DNA uptake

is

closely

of a restriction

investigation

of Health

report

(3,7).

as the possible

of gonococcal

may not occur

influenzae

activity

studies

of genetic

transformation

National

the

for

this

of the Hemophylus

Recent

The specificity acquisition

system

in

seems to resemble

of the DNA receptors

DNA uptake

that

described

DNA uptake

of the specificity

specificity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

natural

by a grant the recipient

that

suggests genetic environment.

from

the

of a

Diseases.

cited

Biswas, G.D., Sox, T., Blackman, E. and Sparling, Bacterial. 129, 983-992. Catlin, B.W. (1967). J. Bacterial. 94, 719-733. Deich, R.A., Sisco, K. and Smith, H.0.(1978). p. 4th European Meeting on Bacterial Transformation University of York, York England. Lacks, S. (1977). p. 35-44 in Portoles, A., Lopez, teds.), Modern Trends in Bacterial Transformation North-Holland Press, Amsterdam. J. Mol. Biol. 3, 208-218. Marmur, J. (1961). Sarubbi, F.A. and Sparling, P.F. (1974). J. Inf. Scocca, J.J., Poland, R.L. and Zoon, K.C. (1974). 369-373. Seto, H. and Tomasx, A. (1974). Proc. Nat. Acad. Sisco, K. and Smith, H.O. (1979) Proc. Natl. Acad. Sparling, P.F. (1966). J. Bacterial. 92, 1364-1371. Sparling, P.F., Biswas, G.D. -and Sox, T.E. (1977). Roberts, R.B. (ed.) The Gonococcus, J. Wiley and Tomasz, A. (1973). p. 81-88 in Archer, L.J. (ed.) Academic Press, New York.

104

P.F.

(1977).

J.

46 Abstracts of the and Transfection, R. and Espinosa, and Transfection,

M.

Dis. 130, 660-663. J. Bacterial. 118, Sci. US 71, 1493-1498. Sci. US, in press. p. 155-176 in Sons, New York. Bacterial Transformation,