Acta Biotechnologica: Volume 10, Number 2 1990 [Reprint 2021 ed.] 9783112581988, 9783112581971

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A c t a ^ H

Volume 10 • 1990- Number 2

BiotedinilDgica Journal of Biotechnology in Industry, Agriculture, Health Care, and Environmental Protection

Akademie-Verlag Berlin ISSN 0138-4988 Acta Biotech noi., Berlin 10 (1990) 2 , 1 0 5 - 212

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Acta Biatachistogiiai Journal of Biotechnology in Industry, Agriculture, Health Care, and Environmental Protection

Volume 10

Edited at the Institute of Biotechnology of the Academy of Sciences of the G.D.R.; Leipzig by M. Ringpfeil, Berlin and D. Pohland, Leipzig

Editorial Board: R. v. Baehr, Berlin A. A. Bajev, Moscow M. E. Beker, Riga S. Fukui, Kyoto P. P. Gray, Kensington I. Y . Hamdan, Kuwait G. Hamer, Zurich L. Herrera, Havana J. Hollo, Budapest

M. V. Ivanov, Moscow D. Meyer, Potsdam A. Moser, Graz P. O. Okonkwo, Enugu G. Pasternak, Berlin W. Scheler, Berlin R. Schulze, Halle B. Sikyta, Prague G. Vetterlein, Leipzig

Managing Editor: L. Dimter, Leipzig

1990 Number 2

A K A D E M I E - V E R L A G - B E R L I N

"Acta Biotechnologica" publishes original papers, short communications, reports and reviews from biotechnology in industry, agriculture, health care and environmental protection. The journal is to promote the establishment of biotechnology as a new and integrated scientific field. The technological character of the journal is guaranteed by the fact t h a t papers on microbiology, biochemistry, chemistry and physics must clearly have technological relevance.

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Acta Biotechnologica Herausgeber: Prof. Dr. Manfred Ringpfeil, Akademie der Wissenschaften der DDR Robert-Rössle-Str. 10, DDR-1115 Berlin-Buch. Prof. Dr. Dieter Pöhland, Institut für Biotechnologie der AdW der DDR J'ermoserstr. 15, DDR - 7050 Leipzig. Verlag: Akademie-Verlag Berlin, Leipziger Str. 3—4, P F 1233, DDR-1086 Berlin; Fernruf: 2236295 und 2236229; Telex-Nr. 114420; Bank: Staatsbank der DDR, Berlin, Konto-Nr.: 6836-26-20712. Cheflektor Zeitschriften: Armin Beck; Redakteur der Abt. Zeitschriften: Cornelia Wanka. Redaktion: Dr. Lothar Dimter (Chefredakteur), Martina Bechstedt, Käthe Geyler, Permoserstr. 15, DDR-7050 Leipzig; Tel. 2392255. Veröffentlicht unter der Lizenznummer 1671 des Presse- und Informationsdienstes der Regierung der.DDR. Gesamtherstellung: VEB Druckhaus „Maxim Gorki", DDR-7400 Altenburg. Erscheinungsweise: Die Zeitschrift „Acta Biotechnologica" erscheint jährlich in einem Band mit 6 Heften. Bezugspreis eines Bandes 210,— DM zuzüglich Versandspesen; Preis je Heft 35,— DM. Der gültige Jahresbezugspreis für die DDR ist der Postzeitungsliste zu entnehmen. Bestellnummer dieses Heftes: 1094/10/2. Urheberrecht: Alle Rechte vorbehalten, insbesondere der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translation into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilm or any other means, without written permission from the publishers. © 1990 by Akademie-Verlag Berlin. Printed in the German Democratic Republic. AN (EDV) 18520 03000

Acta Biotechnol. 10 (1990) 2, 1 0 7 - 1 1 5

Akademie-Verlag Berlin

Significance of Composition and Structure of the Enterobacterial Outer Membrane to the Transport of Desired and Undesired Substances and its Inhibition SELTMANN, G .

Institute of Experimental Epidemiology Burgstraße 37, Wernigerode 3700, G.D.R.

Presented at the 4th Leipziger Biotechnologie-Symposium Dècember 1 2 - 1 6 , 1988

Summary The enterobacterial outer membrane forms a bilayer. Its outer monolayer consists of lipopolysaccharides and proteins, its inner monolayer of phospholipids and proteins. I t thus represents an efficient penetration barrier against hydrophobic and anionic compounds (such as detergents or hydrophobic antibiotics) and against higher molecular substances (such as proteolytic, lipolytic, and murolylic enzymes). Some of the proteins ("porins") form channels through the outer membrane through which neutral and cationic hydrophylic compounds up to a molecular weight of about 800 can pass. Besides the porins additional transport systems have been described. They play an important part in providing the bacteria with substances necessary for their growth, i.e., phosphate, iron ions, and others. Organic polycations are able to generate more or less severe disorganizations in the outer membrane through which they can pass the bilayer ("self-promoted pathway"). Some of these polycations represent efficient antibiotics (polymyxin B , nourseothricin). Bacteria are able to protect themselves against the harmful action of these substances by changing the composition of the outer membrane.

The enterobacterial cell wall consists of two structural units (Fig. 1): K-Antigen

Common Antigen

(ECAj

Lipopolysaccharide (LPS, Endotoxin, 0-Antigen) Outer Membran' Periplasm Space Peptidoglycan '

Fig. 1. Model of the enterobacterial cell wall [2] 1*

Phospholipid Lipoprotein Periplasmic Binding Protein

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Acta Biotechnol. 10 (1990) 2

The first unit is represented by a rigid layer (the so-called peptidoglycan), being responsible for the high mechanical stability of the cell. I£ consists of long chains of the polymer of the disaccharide unit N-acetyl glueosaminyl-/M.4-N-acetyl muramic acid linked /J-1.4 to each other. These polysaccharide chains are cross-linked to each other by peptide bridges of the unit structure L-alanine ->-D-glutamic acid -> m-diaminopimelic acid — alanine. The units are fixed to the carboxyl group of the muramic acid via the amino group of the L-alanine and are bound to each other by a peptide linkage between D-alanine and m-diaminopimelic acid. Thus a high molecular net work of a high mechanical stability arises [1]. The second unit is represented by the outer membrane (Fig. 1). This membrane is unique in nature as the compositions of its inner and its outer monolayers considerably differ from each other. The outer monolayer as main components contains lipopolysaccharides and proteins, the inner monolayer phospholipids and proteins. The chemical structure of the lipopolysaccharides has been outlined in detail elsewhere [2]. Therefore it seems to be sufficient to mention that it represents a typical amphiphile. The hydrophobic moiety, i.e., the so-called lipid A, is an integral part of the outer monolayer while the hydrophilic polysaccharide moiety forms long and heavily coiled chains covering large areas of the cell surface like some kind of bushes. One very easily can imagine that such a combination represents an effective penetration barrier against many environmental substances being more or less harmful to the bacteria. The penetration of hydrophobic compounds will be prevented by the hydrophilic polysaccharide layer, the penetration of hydrophilic compounds by the hydrophobic interior of the outer membrane. A very important feature of the outer monolayer of the outer membrane is the presence of significant amounts of negative charges mainly caused by phosphate and carboxyl groups of the lipopolysaccharides. Additionally the proteins of the outer membrane are known to be negatively charged. As such accumulations of equal charges cause instabilities in the structure of the outer membrane they are neutralized by cations, mainly Ca ++ and Mg ++ . Nevertheless these charges form an effective barrier against the action of negatively charged substances, i. e., the bile acids. However, on the other hand they render sensitive the outer membrane against cationic substances. This will be shown later in detail. It was mentioned above that both monolayers of the outer membrane contain substantial amounts of proteins, namely about 50% (Fig. 1). However, only a few species of proteins have been identified [2]. The bulk of the total proteins, the so-called major proteins, is formed by only 2—5 species. Most of the major proteins are named by the genetic locus by which they are determined. The OmpA protein (present in about 100000 copies per cell, molecular mass of its heat modified form about 33000 daltons) and the outer membrane lipoprotein (molecular mass about 7200 daltons) seem to be important for the cell wall stabilization [2]. On the other hand a group of pore-forming proteins have to be mentioned, in the case of E. coli K-12 named OmpC and OmpF [2]. As bacteria are very fast growing organisms they must transport big quantities of nutrients and waste products through the cell wall. A high percentage of this transport is enabled by these proteins, the so-called porins. The porins are constitutive, i.e., they generally are present in about 100000 copies per bacterial cell. They form hydrophilic water-filled pores which are located across the outer membrane. Neutral and cationic hydrophilic solutes up to a molecular mass of 600—800 daltons diffuse freely, the diffusion of anionic, and in particular of hydrophobic solutes of the same size is retarded. This relative exclusion of hydrophobic and anionic substances seems to be ecological advantageous, as for instance bile acids are anionic and hydrophobic. Besides these constitutive major proteins the synthesis of additional outer membrane

SELTMANN, G . ,

109

Enterobacterial Outer Membrane

proteins under certain limiting conditions can be induced in the bacteria. In such a situation the amount of the protein can become comparable to that of a major protein [2], Most of these proteins are responsible for the penetration through the outer membrane of special solutes. The PhoE protein is synthesized by the bacteria under phosphate limitation in up to 100000 copies per cell. Pores of these proteins are specialized to scavenge even traces of phosphate or phosphate-containing compounds from the medium. PhoE protein is very similar to OmpF protein. I t is, therefore, supposed that the former has evolve^ from a general pore protein. Further proteins being responsible for specific transports through the outer membrane are listed in Table 1. Two of them shall be discussed in detail. Tab. 1. Some minor proteins of the outer membrane of E. coli [1] Protein

mol. wt.

Function

83 K FeuB Cit TonA Cir Bfe LamB Tsx

83000 81000 80500 78000 74000 60000 55000 27000

iron uptake iron uptake iron uptake iron uptake iron uptake vitamine B12 uptake maltose uptake nucleoside uptake

A large group contains the ferric ion transport proteins. Ferric ions are very important for the growth of the bacteria and the development of their virulence. At pH 7 all ferric ions are present in form of the extremely insoluble ferric hydroxide. Therefore, both bacteria and eucaryotes compete for the available iron. Thus the bacteria have developed iron uptake systems in which low molecular chelators (so-called siderophores) and transport systems are integrated [3]. The systems are very complex and also very efficient ones. Several kinds of enterobacterial siderophores are known: ferrichrome, aerobactin, enterochelin, citrate, and coprogen. Very recently in our institute two new siderophores have been discovered, namely colibactin and hafnin [15]. The existence of all these siderophores may reflect the different iron sources to which the enterobacteria are exposed in various environments and also the difficulty to provide them with sufficient amounts of iron. The transport of iron through the outer membrane is an active one, i.e., it needs energy. On the other hand it is well known, that the outer membrane is very poor in available energy. Therefore, models are discussed assuming that there exists an energy flow from the iron transport proteins of the inner membrane directly to those of the outer membrane across the periplasmic space, for instance by a direct contact of both proteins. Another special transport system is connected with the LamB protein, i.e., the receptor protein of phage A [2], This protein plays an important part in the uptake of maltose and maltose oligosaccharides against a concentration gradient up to 100000 [4]. Addition of maltose to the growth medium induces its biosynthesis in amounts comparable to those of the major proteins. LamB pores are penetrable for maltose oligosaccharides up to heptamers, molecular weight about 1250. They are rather, but not absolutely specific. All the transported substances mentioned above are benefit for the bacteria. Above it was stated that the enterobacteria because of the barrier properties of the outer membrane are able to prevent the penetration of many harmful substances present in their environ-

110

Acta Bio techno]. 10 (1990) 2

ment. However, because of the high amount of negative charges present in the outer monolayer of the outer membrane being neutralized by Mg + + or C a + + ions these bacteria are more or less defenceless against the action of chelators and against organic cations, especially polycations. I t has been known since many years [5] that incubation of enterbacteria with EDTA in the presence of T R I S buffer liberates about 50% of the cell wall bound lipopolysaccharides and thus renders the bacteria sensitive against some antibiotics against which they commonly are resistant. I t has been tried to increase the efficiency of antibiotics against enterobacteria by addition of EDTA. In vitro such combinations act excellently, but the disadvantage for in vivo-use results from the fact that EDTA proved to be more harmful towards mammalials than towards the bacteria. Much more important for practise proved to be the action of polycations on the enterobacterial outer membrane. In the following three examples shall be given: the aminoglycoside antibiotics, the polymyxins, and the streptothricins. II

HO

0 Fig. 2. Chemical structure of streptomycin

The aminoglycosides (example: streptomycin, Fig. 2) represent a group of antibiotics. The molecular mass of many of them is lower than 600. That means that they should cross the outer membrane via the porin pores. However, it was found that this pathway is not used throughout. When aminoglycosides are added to the germs, electrostatic binding to anionic sites distributed on the cell surface occurs almost instantaneously [6]. This binding is reversible and concentration dependent. Polycations significantly inhibit the adsorption phase. Both lipopolysaccharides and outer membrane proteins have been found to be the primary target of the aminoglycosides. This phase proved to be energyindependent. After the primary binding the aminoglycosides cross the outer membrane. N A K A E and N A K A E [ 7 ] found that the diffusion rates of this step are higher than had been calculated on the basis of penetration through the porin pores. In the case of P. aeruginosa it was found by HANCOCK et al. [8] that aminoglycosides cross the outer membrane via a self-promoted pathway, involving disruption of lipopolysaccharide-Mg ++ cross bridges. Presumably the action of aminoglycosides on the bridges yields in an at least transient binding to the lipopolysaccharides and a rearrangement of their packing, resulting in the formation of "cracks" in the membrane structure. These artifically induced channels must be hydrophilic to accomodate charged molecules such as aminoglycosides. Very recently R I V E E A et al. [ 9 ] studied the action of aminoglycosides on the cell wall of supersensitive P. aeruginosa mutants. They found that this supersusceptibility is due to alterations in the lipopolysaccharides so that they bind aminoglycosides with a higher affinity. Additionally they stated that the interaction of the aminoglycosides with the cell surface, the disruption of the outer membrane permeability barrier, is an obligate component of cell killing by these antibiotics.

SELTMANN, G., Enterobacterial Outer Membrane

ill

Polymyxin B (Fig. 3) represents a polycationic polypeptide. Two moieties of the molecule can be differentiated: a cyclic head and a linear tail. At the end of the tail a strong hydrophobic residue, i.e., 6-methyl n-heptanoic acid, with its bulky tip is located. The polymyxin B molecule is too big to go through the porin channels, especially because of the cyclic head group. However, because of i t s hydrophobic tip and its many positive charges it very easily enters the outer membrane of enterobacteria and destroys it. V A A R A et al. [10] found that this reaction is much faster than that of the aminoglycosides. To the suspension of S. typhimurium LT2 lysozyme (5 ¡xg/ml) was added. No changes in the optical density at 600 nm (OD6OO) could be detected. After 3 minutes 9 ¡xg/ml polymyxin B were added. Immediately a drastical decrease of OD6OO appeared, two minutes after addition to about 1/3 of the original amount. That means that polymyxin B had caused severe destructions in the outer membrane through which lysozyme could attain and lyze the peptidoglycan layer. leu-dab D-phe

dab

I

I

dab

o thr

^dab^ I dab I •thr

I

dab

F A.

Fig. 3. Chemical structure of polymyxin B

Further evidence that the disorganization of the outer membrane represents the first step in the action of polymyxin was obtained through the study of a polymyxin-resistant mutant of S. typhimurium. The strain was able to resist 100 ¡xg polymyxin/ml. The outer membrane in contrast to the outer membranes of the parent strains did not become permeable to lysozyme upon treatment with polymyxin B [10]. This indicates that the mutation had effected the outer membrane and decreased its susceptibility to the antibiotic. A careful investigation of the lipopolysaccharides of both the parent and the mutant strain indicated four- to sixfold larger amounts of the 4-aminoarabinose and also larger amounts of ethanolamine in the latter [11], That means that at the cell surface of the resistant strain a drastically decreased amount of negative charges is present, thus preventing the primary adsorption process of the polymyxin. The chemical formula of the streptothricins is presented in Fig. 4 [12]. I t shows that the molecule consists of three moieties: the /S-lysine (peptide) chain differing in length between one (streptothricin F) and six (streptothricin A) amino acid residues, the central gulosamine residue, and the so-called streptolidine. Nourseothricin represents a mixture of mainly streptothricin D (45%) and F (45%). The molecule contains several cationic groups, namely the guanidine group in the streptolidine moiety and the amino groups of the /J-lysine(peptide) chain. One of these amino groups is localized at the tip of the peptide chain, perhaps representing the first anchor of the molecule to bind to the negative charges of the bacterial cell surface. After this the antibiotic could be attracted into the cell wall by the other positively charged groups, at last by the rather strong cationic guanidine residue. I t can be seen in Fig. 4 that the head group of the molecule (i.e., the gulosaminyl-streptolidine) is rather bulky. Thus it seems rather unlikely that it is able to pass the outer membrane via the porin pores. Therefore it was logically to assume that nourseothricin passes the outer membrane using a self-promoted pathway similar to those mentioned above. To study this possi-

112

Acta Bio techno!. 10 (1990) 2

o ii

H CH20H N-CH NH M C 0. N—c;. H 2 N-C-0/H ^N-CH CH2 C H h/H H\H CHOH C- - c OH I NH I CO I ch 2 I CH- NH2 l ch 2 I Fig. 4. Chemical structure of nourseoch2 thricin (streptothricin); n varies bet\ fH 2 ween 1 (streptothricin F) and 6 (streptothricin A) nh2 bility indicators were used informing about the degree of disturbances in the outer membrane caused by the action of the antibiotic. The investigations were started using the R-type E. coli strain J 5 3 and the peptidoglycane-destroying enzyme lysozyme (molecular weight about 14,000) as an indicator [13]. The bacteria were suspended in a Ca + + and Mg++ free medium and either lysozyme (20 fig/ml), or nourseothricin (50 fig/ml), or a mixture of both was added. The suspensions were incubated at 37 °C and the optical densities read up to 5 hours (Fig. 5). Lysozyme did not cause any changes in the optical density, nourseothricin only a slight decrease to about 90% after 5 hours. However, the mixture of both causes a significant decrease to about 20% of the original intensity.

Fig. 5. Action of nourseothricin (No), lysozyme (Ly) or a mixture of both on the cell wall of E. coli J 53 [13]

Increasing concentrations of nourseothricin in the range between 5 and 100 ¡xg/ml induced increasing cell lysis. In Fig. 6 the data read after 5 hours incubation are presented. Further increase of the concentration causes the opposite effect: the higher the concentration the less the effect. Concentrations of 2 mg/ml nourseothricin did not cause any lysis by lysozyme. T h a t means t h a t these concentrations stabilize the outer membrane and argues against a mere penetration of the antibiotic through the porin pores. Similar effects of polycations against the enterobacterial cell wall are mentioned in literature [14].

SELTMANN, G . ,

Enterobacterial Outer Membrane

113

Nourseothricin [¡jg/mll Fig. 6. Concentration dependence of the action of nourseothricin on the cell wall of a sensitive ( x — ) and a resistant ( o — ) E. coli R-type strain [13]

Nourseothricin-resistant E. coli strains did not show any of the above-mentioned effects. The effects described above have been found using R-type strains. Strains of this type lack the long coiled polysaccharide chains of the lipopolysaccharides mentioned above, which excellently protect the outer membrane against the harmful action of many substances, for instance bile acids, detergents, or hydrophobic antibiotics. Therefore, it seemed more logically to investigate the naturally occuring S-type strains. As indicators either crystal violet, or Congo red, or the hydrophobic antibiotic novobiocin were used [12]. Congo red is practically not adsorbed by both the resistant and the sensitive strains. After addition of nourseothricin a very fast increase in the adsorption of the dye by both kinds of strains takes place (Fig. 7).

Pig. 7. Effect of nourseothricin on the stainability by Congo red (CR) of sensitive ( x — ) and resistant (•—) strains; o — for comparison: absorption of crystal violet (CV; see Fig. 8). x — CV type l/2a 37° \ . — CV type 1/2 a 0°, 2 b 37° J O — CV type 2b/CV

Acta Biotechnol. 10 (1990) 2

114

Rather different proved to be the absorption of crystal violet (Fig. 8). Nourseothricinresistant strains do not absorb the dye before or after addition of the antibiotic. On the other hand the sensitive strains can be subdivided into two subgroups. Strains of the subgroup 2 a absorb the dye before and after addition of nourseothricin. Strains of subgroup 2 b do not absorb the dye before addition of the antibiotic, however, after addition of nourseothricin the crystal violet absorbing capacity increases drastically within one hour.

100 -

$

80-

Fig. 8. Effect of nourseothricin on the stainability by crystal violet (details see text) 0.5 No addition

1

2 Time

Chi

Novobiocin is an antibiotic to which intact enterobacteria are rather resistant. As can be seen in Fig. 9 addition of sublethal amounts of nourseothricin sensitizes both the sensitive and the resistant strains to novobiocin, however, in the case of the nourseothricin resistant strains the concentration of nourseothricin being necessary to cause this effect is much higher than in the case of the sensitive strains.

Nourseothricin

concentration

i fig /ml ]

Fig. 9. Effect of nourseothricin on the novobiocin sensitivity of a nourseothricin sensitive (R-) and a nourseothricin resistant (R + ) E. coli strain [12] O — 4/1064 ( R - ) x — 4/1064 + novobiocin (2 (j.g/ml) A — 4/1612 (R+) • — 4/1612 + novobiocin

SELTMANN, G., Enterobacterial Outer Membrane

115

The conclusions which can be drawn are: " — I n the first stage of the action of nourseothricin with enterobacteria the antibiotic binds very quickly to superficial parts of the cell wall of both the sensitive and the resistant strains. In this step only very moderate disturbations in the structure of the cell wall are caused, which are indicated by the addition of Congo red. — In the second step nourseothricin enters the outer membrane causing much severe disturbations, which are indicated by the stainability by crystal violet and by the sensitivity against novobiocin. The amount of nourseothricin which is necessary to induce this effect is much lower in the case of nourseothricin sensitive strains than in the case of the resistant strains. We suppose that this difference is caused by differences in the amount of available negative charges in the outer surface of the outer membranes of the sensitive and the resistant strains. Thus it seems logical to assume that a reduced penetrability of the outer membrane plays an important part in the induction of enterobacterial resistance against the antibiotic nourseothricin. References [1] SELTMANN, G.; Die bakterielle Zellwand (1st edition). Jena: Gustav Fischer Verlag, 1982. [2] SELTMANN, G., RIETSCHEL, E. Th.: Wiss. Beitrage Martin-Luther-Univ. Halle —Wittenberg (1988) 3, 77. [3] BRAUN, V., WINKELMANN, G.: Progr. Clin. Biochem. and Med. 5 (1988), 67. [ 4 ] AMES, G . F . - L . : J . B i o e n e r g e t . B i o m e m b r . 2 0 ( 1 9 8 8 ) 1, 1.

[5] LEIVE, L„ SHOVLIN, V. K., MERGENHAGEN, S. E.: J. Biol. Chem. 243 (1968) 6384. [ 6 ] TABER, H . W . , MUELLER, J . P . , MILLER, P . F . , ARROW, A . S . : Microbiol. R e v . 6 1 ( 1 9 8 7 ) 4 , 439. [7] NAKAE, R . , NAKAE, T . : A n t i m i c r o b . A g e n t s C h e m o t h e r . 2 2 ( 1 9 8 2 ) 4 , 5 5 4 .

[8] HANCOCK, R. E . W.: Annu. Rev. Microbiol. 3 8 (1984), 237. [ 9 ] RIVERA, M . , HANCOCK, R . E . W . , SAWYER, J . G . , HAUG, A . , MCGROARTY, E . J . : A n t i m i c r o b .

Agents Chemother. 32 (1988) 5, 649. [ 1 0 ] VAARA, M . , VAARA, T . : A n t i m i c r o b . A g e n t s C h e m o t h e r . 1 9 (1981) 4 , 5 7 8 . [ 1 1 ] VAARA, M . , VAARA, T . , JENSEN, M . , HELANDER, I . , NURMINEN, M., RIETSCHEL, E . T h „ MAKELA, P . H . : F E B S l e t t e r s 1 2 9 ( 1 9 8 1 ) 1, 1 4 5 .

[12] SELTMANN, G.: J . Basic Microbiol. 2 9 (1989) 7, 449. [ 1 3 ] SELTMANN, G . , WOLTER, E . - J . : J . B a s i c Microbiol. 2 7 ( 1 9 8 7 ) 3, 139.

[14] Souzu, H.: Biochim. Biophys. Acta 861 (1986), 361. [15] REISSBRODT, R., RABSCA, W., SPEREITER, J.: Biology of Metals, in press.

Acta Biotechnol. 10 (1990) 2, 116

Akademie-Verlag Berlin

Book Review Joseph FIKSEL, Vincent T.

COVELLO

Safety Assurance for Environmental Introductions of Genetically-Engineered Organisms (NATO ASI Series G: Ecological Sciences, Vol. 18) Berlin, Heidelberg, New York, London, Paris, T o k y o : Springer-Verlag, 1988. 282 pp., DM 1 4 8 , - , I S B N 3-540-18561-5 '

Possible endangering of m a n a n d environment b y biotechnologically employed microorganisms, especially genetically-engineered strains, is intensely discussed in modern biotechnology. This report contains t h e contributions of an international group of scientific a n d regulatory experts who participated in a NATO workshop held in R o m e , I t a l y , J u n e 6 — 10, 1987 t o a special aspect of biotechnology, t h e risk connected with t h e direct introduction of genetically modified microorganisms, especially viruses a n d bacteria, into t h e n a t u r a l environment. Eleven papers deal with general problems of risk analysis a n d assessment, with suitability a n d applicability of t h e available methods, a n d t h e corresponding research needs in biological sciences a n d related disciplines. Regulatory problems a n d perspectives b o t h a t t h e international level a n d in some leading countries in t h e field of biotechnology a r e reviewed b y five f u r t h e r contributions. As s u m m a r y of t h e workshop recommendations are given for a scientific a p p r o a c h t o s a f e t y assurance for environmental introductions of genetically-engineered microorganisms. One of t h e conclusions t h a t genetically-engineered microorganisms c a n n o t be regarded as basically dangerous organisms in comparison with other biotechnologically employed microbial strains is very helpful n o t only in this special field of biotechnology. Risk analysis a n d assessment should be based on t h e investigation of t h e relevant properties of t h e microorganisms a n d their possible relations t o t h e environment a n d n o t on t h e m e t h o d of t h e selection or (if any) t h e genetic optimization. This report should be of interest t o scientific institutions working in t h e corresponding fields of genetics, ecology, biotechnology a n d agriculture as well as to authorities responsible for h e a l t h control, hygiene a n d , especially, for environmental monitoring a n d protection. L . WTTNSOHE

Acta Biotechnol. 10 (1990) 2, 1 1 7 - 1 2 3

Akademie-Verlag Berlin

Outer Membrane Yesiculation of Acinetobacter calcoaceticus BOBNELEIT, P . 1 , BINDEB, H . 2 , KLEBEE, 1

2

H.-P.1

Karl-Marx-Universität Leipzig Sektion Biowissenschaften, Bereich Biochemie Talstraße 33, Leipzig 7010, G.D.R. Karl-Marx-Universität Leipzig Sektion Physik, Bereich Molekularphysik Linnestraße, Leipzig 7010, G.D.R.

Presented at the 4th Leipziger Biotechnologie-Symposium December 1 2 - 1 6 , 1988

Summary For intact cells of A. calcoaceticus 69V susceptibility to hydrophobic agents (antibiotics, dyes) was established. The composition of its outer membrane and comparison with t h a t of a reference strain, A. calcoaceticus COM 5593 with a blocked hydrophobic p a t h w a y , gave no indication of phospholipid bilayer domains as t h e structural basis of these permeability characteristics. The outer membrane composition together with the d a t a of time-resolved fluorescence anisotropy measurements is indicative of a high state of order of t h e hydrocarbon region. A. calcoaceticus 69V releases lipopolysaccharide (LPS)-rich membrane vesicles into t h e growth medium when grown on a hydrophobic carbon source. While t h e cells contain both R-form and S-form LPS, t h e L P S released with t h e vesicles is exclusively of t h e R-type. The same selectivity with respect to L P S composition was observed when L P S was removed from intact cells by EDTANaGl t r e a t m e n t which leads to a break-down of t h e barrier to hydrophobic agents in A. calcoaceticus COM 5593. We propose t h a t due to its physical properties, R-form L P S forms tightly packed structures within t h e membrane which, under certain cbnditions, become destabilized and liberated into t h e surrounding medium. As a consequence, a disturbance of t h e highly ordered lateral molecular arrangement might lead to altered permeability properties of t h e outer membrane as suggested in one of t h e two alternative models existing to explain permeability changes observed in deep rough m u t a n t strains of Enterobacteriaceae.

Introduction By now it has generally been accepted that it is the lipopolysaccharide (LPS) component which mainly contributes to the highly effective permeation barrier function of the outer membrane of gram-negative bacteria to hydrophobic agents. However, despite intensive investigations into the matter still two alternative models exist to explain the differences in the permeability of the outer membrane of wild-type and deep rough LPS mutant strains of Enterobacteriaceae. (i) Deep rough mutants contain phospholipid molecules in the outer laeflet and the phospholipid bilayer domains thus created make a major contribution to the increased permeability, or (ii) A strong LPS-LPS interaction is essential in preventing penetration by hydrophobic solutes in the wild-type, and such a strong lateral interaction is lacking in the altered, deep rough LPS. Thus hydrophobic solutes penetrate through the LPS monolayer domains easily, regardless of the presence or absence of phospholipid bilayer domains [1].

118

Acta Biotechnol. 10 (1990) 2

Here we want to show that at least in Acinetobacter calcoaceticus the formation of the so-called hydrophobic pathway is most probably related to a disturbance of the highly ordered lateral arrangement of LPS molecules in the outer leaflet of the outer membrane. Results and Discussion Most strains of A. calcoaceticus, a member of Neisseriaceae, are able to utilize hydrophobic growth substrates, for instance long-chain hydrocarbons [2], Although much work has been done to elucidate the physical state of the hydrocarbon in the aqueous growth medium, surprisingly the permeability characteristics of the outer membrane of these strains have never been investigated. We have chosen two reference strains: one, A. calcoaceticus 69V shares the common characteristics of this species to utilize n-hexadecane while the other one, A. calcoaceticus CCM 5593, is the only strain we found which . lacks this ability. When compared with wild-type Enterobacteriaceae (Escherichia coli K12 or Salmonella typhimurium LT2) both strains are rather sensitive to hydrophobic antibacterial agents (Tab. 1), but the levels of sensitivity are different. The minimal-inhibitory concentraTab. 1. Minimal-inhibitory concentrations [¡xg/ml] of antibacterial agents for E. coli, 8. typhimurium,, and A. calcoaceticus Agent

Pathway

E. coli

S. typhimurium

A. calcoaceticus

H/h

K12

LT2

69V

CCM 5593

Erythromycin H Rifamycin SV H Gentian violet H

Ra Rb R°

R» Rf Rc

10 20 0.25

R» 60 1.5

Neomycin Gentamycin

7 2.5

7 n.d.

7 2.5

7 2.5

h h

H, hydrophobic; h, hydrophilic; according to [15] R, resistance determined up to 150 ¡xg/mla, 100 ¡xg/mlb, and 2 [xg/ml° n.d., not determined Minimal-inhibitory concentrations were determined in nutrient broth containing different concentrations of the agents tested. The minimal-inhibitory concentration is the lowest concentration which inhibited visible growth after incubation at 30 °C for 24 h with shaking.

tions of the hydrophobic substances tested are higher for A. calcoaceticus CCM 5593 than for 69 V. This is in agreement with the picture we obtained when the uptake of a hydrophobic dye, gentian violet, by intact cells of both strains was measured (Fig. 1). We did not observe gentian violet uptake by CCM 5593, but a definite decrease of dye concentration in the test medium when 69V was used, especially with hexadecanegrown cells. As a first step to shed light on the molecular basis of these permeability properties we analyzed the composition of the isolated outer membranes. The differences found between the three cell types investigated are relatively small, perhaps with the exception of a slight but definite increase in the LPS content of the outer membrane of the CCM 5593 strain (Tab. 2). The most interesting result of this analysis, in our opinion, is the following: The approximate number of LPS fatty acids exceeds the number of phospholipid f a t t y acids by a

Borneleit, P., Binder, H. et al., Outer Membrane Vesiculation

119

Time Cminl Pig. i. Gentian violet uptake by intact cells of A. ealcoaeeticus 69V and CCM 5593 grown on a minimal salts medium with either acetate (10 g/1) or hexadecane (6 g/I) as carbon source Gentian violet uptake was determined at 30 °C, modified after [16]. Tab. 2. Composition of outer membranes of A. calcoaceticus Component A. calcoaceticus 69V Hexadecanegrown Phospholipid (¡¿mol lipid phosphorus/mg protein) [%] LPS ([xmol KDO/mg protein) [%] Protein [%] Molar ratio phospholipid: LPS

Acetategrown

A. calcoaceticus CCM 5593 Acetate-grown

0.16

0.15

0.20

7.6

7.5

7.7

0.090 23.1

0.095 24.0

0.119 40.7

69.3

68.5

51.6

1.9: 1

1.7: 1

2.2: 1

2.0 : 1

2.2 : 1

2.4 : 1

Approximate number LPS-fatty acids: phospholipid fatty acids

KDO, 3-deoxy-D-mora»o-2-octulosonic acid factor of about two. Similar observations had been reported by Gmeiner et al. [3—5] for the L P S mutant strains of 8. typhimurium, but these results could not be confirmed by others [6]. The main point of argument was the question what would happen to the large amount of overproduced L P S , and Gmeiner's suggestion that the L P S molecules become compressed was regarded as unreasonable. However, in a recent paper of Labischinski et al. [7] a surprisingly high state of order of isolated L P S was demonstrated and was suggested to be the crucial factor for the permeation barrier function of L P S in the outer membrane. I n our analysis the molar ratio of phospholipid to L P S is about the same for the three types of outer membranes investigated. This ratio usually is regarded as an important indicator of possible phospholipid bilayer domains. Furthermore, the phospholipid con-

120

Acta Biotechnol. 10 (1990) 2

tents we determined are within the range reported for wild-type Enterobacteriaceae and definitely lower than those of the outer membranes of certain LPS and protein mutant strains where phospholipid bilayer domains have been postulated [6, 8, 9]. Nevertheless, in a previous work [10] we performed one of the typical experiments to test for phospholipids possibly exposed to the outside of the cells. When intact cells were treated with phospholipase A2 no phospholipid degradation was observed for acetategrown cells, but after hexadecane-growth definite amounts of phosphatidylethanolamine and phosphatidylglycerol were degraded. However, the phospholipid amounts susceptible to the phospholipase attack are much too high to be interpreted in terms of the phospholipid bilayer domains postulated. Rather we ascribe this effect to another peculiarity of A. calcoaceticus. It has been described that A. calcoaceticus 69V releases LPS-rich outer membrane vesicles into the culture medium when grown on hexadecane [10, 11]. We could show that this vesicle release occurs over the whole cell surface, and it seems reasonable to assume that this shedding of outer membrane components should affect the outer membrane molecular architecture. In view of the two alternative hypotheses for explaining permeability changes outlined at the beginning of this lecture, it was already suggested by G M E I N E R ' S group [ 4 ] that a loosening of the tight packing of the LPS molecules could account for a susceptibility of phospholipid molecules of the inner membrane leaflet to exogeneously added labeling or degrading agents. It should be noted that the process of vesicle release is more complex than a transient rupture and resealing of the outer membrane because the barrier function to the attack of lysozyme is retained.

1

2

3

4

5

6

7 8 9

Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel after silver staining of LPS. Whole-cell lysates of A. calcoaceticus 69V grown on hexadecane (3 and 6) and acetate (5) and of A. calcoaceticus COM 5593 grown on acetate (4); medium vesicles released from hexadecane-grown 69V (2 and 9); isolated R-form (8) and S-form (7) LPS; the LPS fraction released from hexadecane-grown 69 V by EDTA-NaCl treatment as described for Tab. 3 (1).

B o r n e l e i t , P., B i n d e r , H. et al., Outer Membrane Vesiculation

121

With respect to outer membrane vesiculation we made another interesting observation. Recently we could demonstrate that in addition to the it-type LPS usually described for A. calcoaceticus [12] high-molecular weight S-form LPS is present in the cells (Bobn e l e i t et al., in preparation). However, in the membrane vesicles released into the growth medium exclusively R-form LPS was found (Fig. 2). As far as we know, this is the only example where differences in the proportions of long and short LPS species between cell-bound and released LPS have been reported. Similar LPS compositions as those demonstrated in whole cells have been described both for LPS-vesicles released from normally growing S. typhimurium cells [13] and for the LPS fraction liberated from S. typhimurium by EDTA treatment [14]. Interestingly, both our A. calcoaceticus strains are EDTA-resistant, that is, they do not release LPS or proteins when treated with EDTA. This is in contrast to Enterobacteriaceae which liberate about 50% of their LPS when divalent cations are removed. This different behaviour suggests differences in the anchoring of A. calcoaceticus LPS in comparison with Enterobacteriaceae. I n A. calcoaceticus, LPS could be removed by the combined action of EDTA and high NaCl concentrations. As found in Enterobacteriaceae removal of LPS leads to the disruption of the barrier function of the outer membrane to hydrophobic molecules'(Tab. 3). And interestingly, by EDTA-NaCl treatment too, only the short R-form LPS species are released (Fig. 2, lane 1). Tab. 3. Minimal-inhibitory concentrations (uig/ml) of hydrophobic antibacterial agents for A. calcoaceticus COM 5593 without (control) and after treatment with EDTA-NaCl Agent Erythromycin Rifamycin SV Novobiocin Gentian violet Sodium deoxycholate

Control
10"

> 108

55

6 - 104

98

100

97

97

98

50

55

90

0

75

Passage

5

200

Medium

906

906

T906

902

T902

1

+

T902

++++

^

Stamm 383

Kapselbldg. Virulenz (LD 50 ) Immunogenität (Tot-Vakzine, % Überlebende) Immunogenität (KE, % Überlebende)

+/+ +

+/ + +

+

++

+

1

14

24

> 108

4

83

25

33

82

97

53

13

0

15

25

Diese Ergebnisse lassen erkennen, daß zwischen der Größe der Kapsel, der Virulenz und der Immunogenität keine direkte Korrelation besteht. Bei den geprüften Beispielen hängt dieses Verhalten wesentlich von den Ausgangsstämmen sowie von den Kultivierungsbedingungen ab. Im weiteren Verlauf der Untersuchungen prüften wir mittels SDS-PAGE die Zusammensetzung von Kapselextrakt- und OMP-Material hinsichtlich ihrer Beeinflußbarkeit durch die Kultivierungsbedingungen. Im Kapselextraktmaterial wurde bei beiden Stämmen LPS mit vorwiegend R-Charakter nachgewiesen (Abb. 3 und 4). In Abhängigkeit von den Kultivierungsbedingungen traten deutliche Veränderungen in der LPS-Struktur auf.- Höhermolekulare Anteile (Abb. 3, Pos. 1 und Abb. 4, Pos. 1) nahmen ab, während im Bereich niederer Molekülmassen ( < 12 kDa) zusätzlich auftretende Banden die Heterogenität der LPS-Strukturen erkennen ließen (Abb. 3, Pos. 4 und 5; Abb. 4, Pos. 2, 3 und 5). Diese Strukturveränderungen könnten die Ursache für ein bisweilen verändertes serologisches Verhalten mancher Stämme bei längerer Laborhaltung sein. Die OMP-Profile beider Stämme zeigen Hauptbanden mit Molekülmassen von 39 kDa (PI) und 25 kDa (P3) (Abb. 5 und 6). Der Stamm 383 besitzt ein weiteres Protein von 33 kDa (P2), das bei Stamm 880 lediglich nach Passagierung im Eisen-Normalmedium bei 27°C auftritt (Abb. 5, Pos. 4 und 7). Zur Aufklärung der Funktion dieser Proteine der äußeren Membran sind weitere Untersuchungen erforderlich.

Rosnek, H., Rohrmann, B. u. a., Oberflächenstruktur von Pasteurella l. i..

multocida

......SUW.;.

1

2

3

4

5

Abb. 3. SDS-PAGE von Kapselextraktmaterial des Stammes 880 Medium 696: chemisch definiertes Medium 906 mit 5 % Hefezusatz 1 - 880/696/4 K E 40 ¡ig 2 - 880/906/150 K E 50 (ig 3 - 880/902/160 K E 40 ¡ig 4 - T 880/902/165 K E 50 ¡ig 5 - T 880/906/160 K E 40 ¡ig

I rne» 17500 12500

1

2

3

.

5

Abb. 4. SDS-PAGE von Kapselextraktmaterial des Stammes 383 1 - 383/696/4 K E 10 ¡ig 2 - 383/906/200 K E 10 ¡ig 3 - 383/902/380 K E 10 ¡ig 4 - T 383/906/240 K E 10 (ig 5 - T 383/902/240 K E 10 (ig

148

Acta Biotechnol. 10 (1990) 2

66000 -5000 -J?1 -P2

-1-3

25000

\h20Q 12500

3

4 - 5 6 7

1 2 3 4 5 6 7

— Bichproteine 880/696/3 OMP/Ü 10 880/906/271 OMP/Ü 40 - T 880/906/257 OMP/Ü 40 880/696/3 OMP 60 880/906/271 OMP 60 - T 880/906/257 OMP 100

1 2 3 4 5 6 7

383/906/3 383/906/224 - T 383/906/360 383/906/3 383/906/224 - T 383/906/360 — Eichproteine

(ig ¡ig (ig ¡ig (ig (ig

Abb. 5. S D S - P A G E von OMP-Material des Stammes 880 (Ü: Überstand nach Sarkosylextraktion)

il r2

-

t-3

-

4-5000 4ÖOOO

^ ^Ü

25000;

1-J200 Iii 5 0 0

2

3

5 6

7

OMP OMP OMP OMP/Ü OMP/Ü OMP/Ü

80 80 80 10 10 10

¡ig ¡ig (ig ¡ig ¡ig ¡ig

Abb. 6. S D S - P A G E von OMP-Material des Stammes 383 (Ü: Überstand nach Sarkosylextraktion)

Für die technische Unterstützung bedanken wir uns ganz herzlich bei Frau Monika GODAT und Frau Gisela THIELE.

ROSNER, H., ROHRMANN, B. U. a., Oberflächenstruktur von Pasteurella multocida

149

Literatur [ 1 ] FLOSSMANN, K . D . , R O S N E R , H . , GRTTNKE, U . , MIOSGA, N . : Z . a l l g . M i k r o b i o l . 2 4 ( 1 9 8 4 ) , 2 3 1 . [ 2 ] R E B E R S , P . A . , HBDDLESTON, K . L . , RHOADES, K . R . : J . B a c t e r i o l . 9 3 ( 1 9 6 7 ) , 7 . [3] ACHTMAN, M., S C H W I C H O W , S., H E L M U T H , R., MORELLI, S., MANNING, P. A.: Mol. Gen. Genet.

164 (1978), 171.

[ 4 ] R E E D , L . T „ MTJENOH, H . : A m . J . H y g . 2 7 ( 1 9 3 8 ) , 4 9 3 . [5] CAMERON, C. M., ENGELBRECHT, M. M., VERMEXTLEN, A.

S. M. : Onderstepoort J . Vet. Res. 45 (1978), 215. [6] DREWS, G. : Mikrobiol. Praktikum für Naturwissenschaftler. Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Verlag, 1968. [7] GÜNTHER, H., R O S N E R , H., GODAT, M., E R L E R , W. : Acta histochemica, Suppl.-Band XXXIII (1986), 293.

[8] HITCHCOCK, P . J . , BROWN, T. M.: J . Bacteriol. 154 (1983), 269.

Acta Biotechnol. 10 (1990) 2, 150

Akademie-Verlag Berlin

Book Review Wulf

CBUEGER,

Anneliese

CRUEGER

Biotechnologie — Lehrbuch der angewandten Mikrobiologie Dritte, neubearbeitete Auflage München, Wien: R. Oldenbourg Verlag, 1989 342 S„ 224 Abb., 342 Tab., DM 7 6 , - , ISBN 3-486-28403-7

Microbiology is gaining in important in the production of enzymes and chemicals. A lot of applications in basic research, in diagnostics, in agriculture and in the area of environment become apparent. The well-proven textbook of microbiology in its third edition shows the newest position of research. Authors describe the characteristics of the microorganisms and microbiological and techniques important in praxis. The book is intended for lectures and advanced students of microbiology. I t will also serve as a useful reference book for experts in pharmaceutical and biotechnological research.

Acta Biotechnol. 10 (1990) 2, 1 5 1 - 1 6 1

Akademie-Verlag Berlin

Adhesins of Escherichia coli NIMMICH, W .

Wilhelm-Pieck-Universität Rostock Bereich Medizin Institut für Medizinische Mikrobiologie Leninällee 70, Rostock 2500, DDR

Presented at the 4th Leipziger Biotechnologie-Symposium December 1 2 - 1 6 , 1988 Summary E. coli has got increasing importance as a causative agent of intestinal and extra-intestinal diseases. In both these infections adhesion of the bacteria to mucous surface cells are initial events for colonization and development of infection. Adhesins are bacterial recognition proteins which specifically interact with carbohydrate moieties of glycoproteins or glycolipids on mammalian cells. The adhesiveness of bacteria is associated with filamentous surface appendages, designated as fimbriae or pili, as well as with non-fimbrial components. Some recent data on the nomenclature, classification, disease association, receptor specificity, and topographic arrangement are presented. The correlation between E. coli O : K : H serovar and fimbrial antigens is demonstrated on the basis of E. coli isolated from patients with urinary tract infections. Hitherto unknown non-fimbrial adhesins are briefly described.

Introduction The pathogenic potential of E. coli has been attributed to a complex of virulence factors such as defined 0 and K antigens, enterotoxins, haemolysin, iron uptake systems, serum resistance, and especially the adhesins. These mediate the bacterial adherence to epithelial cells and have been made responsible for the initial step in the development of an infectious process. Adhesins are bacterial proteins which recognize a carbohydrate moiety of glycoproteins or glycolipids on eucaryotic cells, a sterochemical interaction between complementary molecules that may be portrayed as a lock-in-key mechanism a t the molecular level. Nomenclature T h e capacity of E. coli to agglutinate erythrocytes led to the detection of adhesive fac-

tors. The haem^igglutinins responsible for these reactions were found to be associated with the presence of non-flagellar filamentous appendages protruding from the bacterial surface and were designated fimbriae [1]. A few years later the same structures were named pili [2]. Both terms are used as synonyms, though the designation pili was proposed to be reserved for the structures involved in conjugation [3]. Recently Another term fibrils was coined for very thin filaments, the identification of which probably depends on the resolving power of the electron microscop used [4]. The term adhesin

152

Acta Biotechnol. 10 (1990) 2

was introduced as a more comprehensive one since adherence was observed also to other than red blood cells [5]. Several other designations have been used in the literature for adhesive components of E. coli from different sources which briefly should be mentioned here. Fimbrial adhesins detected among human enterotoxigenic E. coli have been described as "colonization factor antigens" (CFA I, II), "coli surface antigens" (CS1—CS6), and "putative colonization factors" (PCF) [6, 7, 8]. Adhesive factors in animal enterotoxigenic E. coli were regarded as capsular antigens (K88, K99) [9,10], and some were termed according to the strain number (987 P, F41) [11, 12]. For the fimbrial adhesins of uropathogenic E. coli a nomenclature was introduced based on serologically established fimbrial F antigens [13]. But the designation "pyelonephritis associated pili" (PAP) has also been used [14], Recently adhesins have been termed according to their receptor specificity which will be delt with separately. Classification and Characterization Though fimbriae differ in morphology (e.g. in diameter between 2 and 7 nm) as observed by electron microscopy this has not been adopted for use in routine. The haemagglutinating activity of E. coli with erythrocytes of various animal species was used to distinguish between adhesive strains by their different haemagglutination (HA) patterns [1, 15]. But the method has not found wide applicability in practice. One main observation, however, is still of importance to-day. That is the finding that the HA may be inhibited by D-mannose or its derivatives. This mannose-sensitive (MS) HA of guinea pig erythrocytes proved to be useful for the detection of so-called common or type^l fimbriate E. coli [15]. A HA that is not affected by mannose has been called mannoseresistant (MR) and the adhesins MR adhesins, respectively. E. coli carrying MR adhesins are associated with disease. In several cases no fimbriae could be detected in adhesive strains by electron microscopic investigation. Thus fimbrial and non-fimbrial adhesins have to be considered [15], Chemical analyses including amino acid composition or sequencial studies as far as highly purified samples are available were also applied successfully to characterize adhesins [16, 17, 18]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) proved to be useful since each adhesin can be characterized by a single peptide band of special molecular size [19]. Furthermore the receptor specificity gained increasing importance as classification principle, but adhesins exhibiting the same specificity may be different in the subunit size and serological properties [13, 18, 20]. The serological classification of fimbrial adhesins has been reviewed very recently [20]. Disease Association Fimbriate E. coli are able to cause intestinal and extraintestinal diseases (Tab. 1). Intestinal infections in man are due to E. coli carrying colonization factor antigens (CFA) which confer specific adhesion properties on the bacteria to the human intestinal epithelium. The actual diarrhoea is produced by elaboration of enterotoxins. The same mechanism is true for diarrhoea in piglets (K88, 987P) and in calves (K99, F41) [21], E. coli strains with fimbrial antigens F7 through F16 are responsible for urinary tract infections (UTI) [22]. For type 1 fimbriate E. coli no convincing evidence has been established as being involved in the development of human disease. In the second column of Tab. 1 a simplified nomenclature using the term F antigen instead of the original designation is shown [13].

NIMMICH, W . ,

Adhesins

153

Tab. 1. Original and renamed fimbrial adhesins and disease association. Type I CFA I CFA II PCF8775 K88 K99 987P F41

FI F2 F3 —

F4 F5 F6 —

F7-F16

î diarrhoea diarrhoea diarrhoea diarrhoea diarrhoea diarrhoea diarrhoea UTI

no man man man pig pig, calf, Pig calf man

Correlation to E. coli 0, K, H Antigens From the vast diversity of possible 0 : K : H serovars — resulting from the combination of 171 somatic 0 , 73 different capsular (K) and 56 different flagellar (H) antigens — only a limited number of serovars are predominant in and known to be related to disease. The association between adhesins and certain O serogroups in enterotoxigenic E. coli from human and animal diarrhoea has been reviewed [16, 21]. A close association was found between fimbrial F antigens and well defined 0 : K : H serovars among urinary E. coli isolates causing MRHA of human erythrocytes. Some of the firmly established correlations are the following: F7 t ,t and 0 6 : K2 : H I , F8 and 018 : K5 : H - , F9 and 0 1 : K l : H - , F10 and 0 7 : K l : H~, F l l and 0 1 : K l : H7, F12 and 016 : K l : H6 [13, 22, 23]. An 0 4 : K12 : H5 strain was found to harbour 4 different fimbrial types: F13, F14, F16 and F1C [19, 20].

Receptor Specificity The serologically different fimbrial antigens F7 through F16 just mentioned above as associated with UTI all were found to exhibit the same binding specificity " P " (Tab. 2) [22]. These, therefore also termed P fimbriae, recognize the disaccharide L oi io •© »H -Í-H Í-H X X X I M I CO ^ I

e É» «o;

O A O ft Cb A i i i i i i o o o o o o A A A A A A

•a o •ë m o M 's I t^ff I

m

¡a -Ö 3

a

a N

H

oí

a 03

•«r -a o tí '3 ¡s

-O tí I £

B

S® m O

-h œ io io cica H« n ^ o o a5 « >1 H H —————— ••H©i HoT H o rot rot r tor to 0 r. »i>{ Ot-t O t00 00 00 z z Z Z Z Z Z «

tí

10®0 4-+> ® -4t¡o-r

KASPRZAK,

H. (

R E H A K , E . U.

a., Bordetella bronchiseptica Mutantenstämme

189

und lyophilisiert. Die Lyophilisate (5 mg) wurden in einem Probenpuffer (0.04 m Tris/HAc pH 7,4, 2% SDS, 5% Merkaptoethanol) aufgenommen und 5 min auf 100 °C erhitzt. Pro Depot wurden 40 ¡xl Lösung aufgetragen. Die Färbung der Proteine erfolgte mit Coomassiebrillantblau G-250).

Ergebnisse und Diskussion Ziel der Transposonmutagenese von B. bronchiseptica ist es, potentielle Impfstoffmutan ten zu entwickeln, die gegenüber dem Wildstamm über eine abgeschwächte Virulenz verfügen und sich durch definierte Eigenschaften (Auxotrophiemarke) eindeutig von diesen unterscheiden lassen. Daher wurden die Transposonmutanten im Vergleich mit ihren Wildstämmen hinsichtlich ihrer biologischen Parameter in vitro und in vivo getestet. Die Ergebnisse sind in den Tabellen 2 Und 3 dargestellt. Tab. 3. Vergleich der Polypeptidmuster von Wildstämmen und den dazugehörigen Transposonmutanten Stämme 873

MW (KD) 144 ± 6 134 ± 5 133 ± 5 124 ± 5 123 ± 5 103—118 ± 5 98 ± 4 109 ± 5

—

+ + + +

873 873 : : Tn5/1 ::Tn5/9

+ -

N104 T Ratte ::Tn5/4

Ratte : :Tn5/l

+

+

+

+

+

+

—

—

—

—

—

—

—

+ +

+ + + +

—

+

+

—

—

—

—

N 104 N104 ::Tn5/3 ::Tn5/6

—

+ + + +

—

+ +

N104

•

+ —

+ +

+ +

—

— —

+ +

+

+

—

—

—

—

—

—

—

—

+

—

—

+

+

+

Die Stämme Ratte und 873 befinden sich vor der Mutagenese im Phase-I-Zustand, während der Stamm N 104 ein Phase-III-Stamm ist. Nach erfolgter Mutagenese befinden sich alle Stämme im Phase-III-Zustand. Das wird durch unsere Versuchsergebnisse belegt: 1. Phase-I-Stämme bewirken auf BOBDET-GENGOU-Agar eine /S-Hämolyse, die entsprechenden Mutanten nicht. 2. Die Kapselfaktoren 8, 10 und 11 sind nur bei Phase-I-Stämmen in ihrer Gesamtheit nachzuweisen, während die Mutanten der Phase-I-Stämme nur noch Faktor 8 exprimieren. 3. Nur die Phase-I-Stämme agglutinieren Erythrozyten von Pferd und Kalb, Phase-IIIStämme und die Mutanten zeigen nur eine geringe bzw. keine Hämagglutination. 4. Die Kolonisationsdauer im Meerschweinchen ist bei den Wildstämmen der Phase I länger als die des Wildstammes N 104 (Phase I I I ) und der Mutanten. 5. Es findet keine Bildung des hitzelabilen Toxins (DNT) bei dem Wildstamm in Phase I I I und bei den Mutanten statt. 6. Die Proteinprofile der SDS-PAGE der Mutanten zeigen das charakteristische Bild von Phase-III-Stämmen (Abb. 1, Tab. 3). 7. Schutzversuche an Mäusen und Meerschweinchen fielen negativ aus.

190

Acta Biotechnol. 10 (1990) 2

t

2

*

4

-Hl

8

T

8

9

Abb. 1. PAGE-Elektropherogramm von Bordetella ferowcAise^iica-Wildstämmen und den entsprechenden Transposonmutanten 1 6 -

873; 2 - 873: : T n 5 [1]; 3 - 873: : T n 5 [9]; 4 - N 104; 5 - N 1 0 4 : : T n 5 [3] N~104: :Tn 5 [6]; 7 - N 104: :Tn 5 [4]; 8 - Ratte; 9 — Ratte: :Tn 5 [1]

Auch zahlreiche nachfolgende Transposonmutagenesen mit anderen Plasmiden verschiedener Inkompatibilitätsgruppen und dem Transposon Tn5 führten nie zu virulenten Transkonjuganten im Phase-I-Zustand. B. bronchiseptica-Stämme werden in der Literatur bis auf zwei Ausnahmen als plasmidfrei beschrieben. Daher liegt die Vermutung nahe, daß die Plasmidübertragung in virulente Wildstämme nur unter bestimmten Bedingungen möglich ist, die in vivo und bis jetzt in vitro nicht vorliegen. Es ist anzunehmen, daß die Rezeption des Pilus all die Lipopolysaccharid-Membranproteine (Rezeptoren) der Zellwand der virulenten Stämme nicht stattfinden kann. Da virulente Stämme eine hydrophobere Oberfläche als avirulente Stämme haben, besteht die Möglichkeit, daß die Rezeptoren durch zusätzliche Oberflächenstrukturen (Kapsel) verdeckt werden. Die B. bronchiseptica-FeXdsta.mmG sind sehr phasenlabil. Durch eine Mutation im vir Gen kann die Expression der Virulenzfaktoren abgeschaltet werden, so daß Phase-IIIStämme entstehen [8]. Die phänotypischen Veränderungen des Phasenwechsels können in vitro als reversible Antigenmodulation nachvollzogen werden (Nährmedien, Herabsetzen der Temperatur). Zur Zeit wird deshalb versucht, mit Hilfe dieser Antigenmodulation kurzzeitig Phase I-Stämme so zu modulieren, daß auf diese Weise durch die veränderte Oberflächenstruktur Plasmide in virulente Bordetellenstämme übertragen werden können.

Literatur [1] WÜNSCH, U . : Untersuchungen zur Entwicklung von Bordetella 6roracAisej)

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die Raumbelastung 1464 g CSB/m 3 • Tag, was umgerechnet auf eine Einheit der Belebtschlammbiomasse einen Wert von 0,5 kg CSB/kg organische Trockenmasse • Tag ergab. Der prozentuelle Beseitigungseffekt der Abwasserverunreinigungen betrug durchschnittlich 79% und war im Bereich von 38%—95% enthalten, der mittlere Schlammindex betrug 301 cm 3 /g organische Trockenmasse. Die dargestellten Größen zeigen, daß der im Modellsystem erfolgende Prozeß als konventionelles Belebtschlammverfahren zu qualifizieren ist. Während der Untersuchungsdauer hat der Belebtschlamm eine immer schlechtere Senkbarkeit aufgewiesen, was zur Verringerung seiner Konzentration im Bioreaktor führte und schließlich einen gleichmäßigen Anstieg der Schlammbelastung verursachte. Korrelation zwischen den Messungen der metabolischen und der Intensität des Belebtschlammverfahrens

Mikroorganismenaktivität

Die in Tab. 2 zusammengestellten Untersuchungsergebnisse dienten zur Festlegung der Korrelation der einzelnen Parameter des Belebtschlammverfahrens. Diese Parameter, d. h., die metabolische Aktivität der Mikroorganismen und die Eliminationsleistung wurden in drei verschiedenen Einheiten ausgedrückt. Neben den Werten der Mikroorganismenaktivität und der Eliminationsleistung auf eine Volumeneinheit der Belüftungskammer wurden sie auf eine Einheit der Mikroorganismenbiomasse umgerechnet, wobei angenommen wird, daß der Richtwert hierfür die organische Belebtschlammtrockenmasse und der ATP-Gehalt in den Mikroorganismenzellen ist. Die letzte Größe, d. i. der ATP-Gehalt im Belebtschlamm wurde gleichzeitig als Richtwert der metabolischen Aktivität angesehen. Das obige berücksichtigend, bemühte man sich zu beurteilen, bis zu welchem Grade die Eliminationsleistung im Bioreaktor von den Messungen der metabolischen Mikroorganismenaktivität abhängt, indem man den Korrelationskoeffizient bestimmt und die betrachtete Wechselbeziehung durch entsprechende mathematische Gleichungen beschrieben hat (Tab. 3). Da ATP-Messungen nicht so oft durchgeführt wurden wie die Bestimmungen der Atmungs- und Dehydrogenaseaktivität, hat man bei den Berechnungen lediglich diese Versuchstage berücksichtigt, an denen sämtliche Bestimmuligen ausgeführt wurden, damit die unterschiedliche Messungshäufigkeit die zu bestimmende Korrelation nicht entstellt (n = 19). Die erreichten Ergebnisse weisen darauf hin, daß man bei Messungen der Atmungsaktivität eine sehr gute Korrelation erhalten hat. Für die auf eine Volumeneinheit bezogene Eliminationsleistung und Atmungsaktivität betrug der Korrelationskoeffizient 0,80 und stieg noch ein wenig bis 0,82 bei Angabe dieser Werte auf 1 Gramm Belebtschlammtrockenmasse, um bei der Umrechnung auf ATP den höchsten Wert aufzuweisen (r = 0,96). Bei Messungen der Dehydrogenaseaktivität hat man sowohl für dife auf eine 'Volumeneinheit als auch auf 1 Gramm Trockenmasse ausgedrückten Parameter eine kaum gemäßigte Korrelation erreicht (Tab. 3). Eine sehr hohe Korrelation wurde dagegen erreicht, wenn man sowohl die Eliminationsleistung als auch die Dehydrogenasenaktivität auf den ATP-Gehalt in den Mikroorganismen bezogen hat. Einen großen Einfluß auf die berechnete Korrelation haben die am 39. Versuchstag erreichten Ergebnisse gehabt. Dies sieht man sowohl bei den Messungen der Atmungsaktivität, als auch bei denen der Dehydrogenaseaktivität. Eine Analyse der in Tab. 2 zusammengestellten Ergebnisse läßt vermuten, daß es die plötzliche Herabsetzung der Schlammbelastung an diesem Tag verursacht hat. Gewiß haben also Mikroorganismen dank ihrer homeostatischen Eigenschaften den Energiebedarf vom Reservestoff gedeckt, und dank dessen ihre vorhergehende Aktivität beibehalten. Wenn man die Ergebnisse vom 39. Versuchstag unberücksichtigt läßt, da erreicht man einen Anstieg

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