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Der Umgang mit Mikroorganismen ist in Deutschland durch ein dichtes Netz von Gesetzen und Verordnungen geregelt. Die fur das MIKROBIOLOGISCHE PRAKTIKUM relevanten Bestimmungen werden im Folgenden erlautert. In anderen Staaten (z. B. Osterreich oder Schweiz) gelten andere Gesetze und Vorschriften.

A novel Sphingomonas strain was isolated from a sample of desert soil collected near Jeddah in Saudi Arabia. A polyphasic approach was performed to characterize this strain, initially designated as G39T. Cells of strain G39T are motile, Gram-negative, catalase- and oxidase-positive. The strain is able to grow aerobically at 20-35 °C, pH 6.5-8 and tolerates up to 4 % (w/v) NaCl. Based on 16S rRNA gene sequence similarity, the closest relative type strains of G39T are Sphingomonas mucosissima DSM 17494T (98.6 %), S. dokdonensis DSM 21029T (98.4 %) and S. hankookensis DSM 23329T (97.4 %). Furthermore, the average nucleotide identities between the draft genome sequence of strain G39T and the genome sequences of all other available and related Sphingomonas species are significantly below the threshold of 94 %. The G+C content of the draft genome (3.12 Mbp) is 65.84 %. The prevalent (>5 %) cellular fatty acids of G39T were C18 : 1ω7c, C16 : 1ω7c and/or C16 : 1ω6c, C14 : 0 2-OH and C16 : 0. The only detectable respiratory quinone was ubiquinone-10 and the polar lipids profile is composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, as well as unidentified lipids, phospholipids and glycolipids. The results of the conducted polyphasic approach confirmed that this isolate represents a novel species of the genus Sphingomonas, for which the name Sphingomonas jeddahensis sp. nov. is proposed. The type strain of this species is G39T (=DSM 103790T=LMG 29955T). peerReviewed

Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated. One isolate was taxonomically affiliated asPseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described. CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix. The mature form of the inducible enzyme consisted of one type of subunit withM r = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (β-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression inEscherichia coli, and its nucleotide sequence was determined. The enzyme exhibited only 27–28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue. In addition, the strong inhibition of the enzyme by Pefabloc® and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of β-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism.

All publicly accessible microbial genome databases were searched for the occurrence of genes encoding proteins homologous to the cyanophycin synthetase (CphA) of Synechocystis sp. strain PCC 6803 in order to reveal the capability of microorganisms not belonging to the cyanobacteria to synthesize cyanophycin. Among 65 genome sequences, genes homologous to cphA were found in Acinetobacter sp. strain ADP1 (encoding a protein homologous to CphA with 40% amino acid identity), Bordetella bronchiseptica strain RB50 (39%), Bordetella pertussis strain Tohama I (39%), Bordetella parapertussis strain 12822 (39%), Clostridium botulinum strain ATCC 3502 (39%), Desulfitobacterium hafniense strain DCB-2 (38%) and Nitrosomonas europaea strain ATCC 25978 (37%). The gene homologous to cphA from Acinetobacter sp. strain DSM 587 was amplified by PCR, ligated to the vector pBluescript SK(-) downstream of the lac promoter and introduced into Escherichia coli. The recombinant strain of E. coli expressed CphA activity at up to 1.2 U/mg protein and accumulated cyanophycin to up to 7.5% of the cellular dry matter, indicating that CphA of Acinetobacter sp. strain DSM 587 is functionally active. In Acinetobacter sp. strain DSM 587 itself, cyanophycin accumulated to up to 1.4% of the total protein under phosphate-limited conditions, and cyanophycin synthetase activity was detected, which indicated the function of cyanophycin as a storage compound in this strain.

Microorganisms are able to synthesize three different polyamides by enzymatic processes independently from ribosomal protein biosynthesis: poly(gamma-D-glutamic acid), poly(epsilon-L-lysine) and multi-L-arginyl-poly(L-aspartic acid) which is also referred to as cyanophycin. These polyamides, which occur mainly in Bacillus spp. (and only a few other eubacteria and the nematocysts of Cnidaria, in Streptomyces albulus or in cyanobacteria, respectively), have recently attracted considerable interest of the chemical industry and may be suitable for various applications. This review summarizes our current knowledge on the occurrence, biosynthesis, physiological functions, and biodegradation as well as on the properties and putative applications of these polyamides. Emphasis is placed on the enzymology of the polymerization and on the genes encoding the polymerizing enzymes, which have only recently become available for cyanophycin synthetases. Prospects for novel production processes. in particular for cyanophycin, are also presented.

The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase ( cphA ) and cyanophycinase ( cphB ), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-β- d -thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50°C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, l -arginine and l -aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, α-arginyl aspartic acid dipeptide, and poly-α,β- dl -aspartic acid as primers and also incorporated β-hydroxyaspartic acid instead of l -aspartic acid or l -canavanine instead of l -arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.

Poly(γ- d -glutamic acid) (PGA)-producing strains of Bacillus species were investigated to determine their ability to contribute to reducing the amount of ammonium nitrogen in liquid manures and their ability to convert some of the ammonium into this polyamino acid as a transient depot for nitrogen. Organisms that do these things should help solve the serious environmental problems which are caused by the use of large amounts of liquid manure resulting from intensified agriculture; these problems are mainly due to the high content of ammonium nitrogen. Bacillus licheniformis ATCC 9945 and Bacillus subtilis were able to grow in liquid manure and to produce PGA in the presence of sodium gluconate. On artificial liquid manure these two strains were able to produce 0.85 and 0.79 g of PGA per liter, respectively. Under conditions that are found in intensified farming situations the ammonia content was reduced within 48 h from 1.3 to 0.75 g/liter. One mutant of B. subtilis 1551 impaired in the catabolism of PGA was obtained after nitrosoguanidine mutagenesis. This mutant produced PGA at a final concentration of 4.8 g/liter, whereas the wild type produced only 3.7 g/liter.

A recent study indicated that Bacillus subtilis catabolizes acetoin by enzymes encoded by the acu gene cluster (F. J. Grundy, D. A. Waters, T. Y. Takova, and T. M. Henkin, Mol. Microbiol. 10:259–271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by aco gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth. By hybridization with a DNA probe covering acoA and acoB of the AoDH ES from Clostridium magnum , genomic fragments from B. subtilis harboring acoA , acoB , acoC , acoL , and acoR homologous genes were identified, and some of them were functionally expressed in E. coli . Furthermore, acoA was inactivated in B. subtilis by disruptive mutagenesis; these mutants were impaired to express PP i -dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source. Therefore, acetoin is catabolized in B. subtilis by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described acu genes obscure.

In der nachfolgenden Tabelle (◉ Tab. 7.1) sind samtliche im Mikrobiologischen Praktikum beschriebenen Versuche, Exkursionen und Demonstrationen aufgefuhrt. Daneben sind acht Zielgruppen bzw. Ausbildungsrichtungen aufgefuhrt, denen Versuche zugeordnet sind, die uns fur die jeweilige Zielgruppe bzw. Ausbildungsrichtung besonders geeignet erscheinen (✓). Die Abschatzung der Durchfuhrbarkeit basiert auf Erfahrungswerten und berucksichtigt die fur die Versuche notwendigen Geratschaften sowie den Kenntnisstand der Teilnehmer.

Zu Beginn des Mikrobiologischen Praktikums sollen einige einfuhrende Anmerkungen gemacht werden, welche die in jeglicher Hinsicht auserordentlich grose Diversitat der Mikroorganismen verdeutlichen und einen Uberblick uber die verschiedenen Gruppen der Mikroorganismen und ihre Lebensweisen geben. Die letzte offizielle Bilanzierung des taxonomischen Standardwerkes Bergey’s Manual of Systematic Bacteriology aus dem Jahre 2003 wies exakt 6466 validierte Spezies aus, beim Schreiben dieser Auflage ist die Zahl auf rund 9000 gestiegen (angegeben in der List of Prokaryotic Names with Standing in Nomenclature, http://www.bacterio.cict.fr ). Bakterien zeichnen sich durch eine ungewohnlich grose Vielfalt aus. Entgegen haufig getatigten Auserungen betrifft dies sogar die Zellmorphologie und die Zellgrose. Besonders vielseitig ist der Stoffwechsel der Bakterien. Hier gibt es zahlreiche Stoffwechselleistungen, die ausschlieslich von Prokaryonten katalysiert werden konnen (Monopole der Prokaryonten) und ohne die ein Leben auf unserem Planeten auf lange Sicht nicht moglich ware. Diese kurzen Ausfuhrungen konnen die Lekture von Lehrbuchern der Mikrobiologie nicht ersetzen; sie sollen jedoch auf das Mikrobiologischen Praktikums einstimmen und einige besondere Aspekte beim Umgang mit Mikroorganismen hervorheben.

Die unten aufgefuhrte Liste der Medien, die im Mikrobiologischen Praktikum Verwendung finden, verzichtet zugunsten der Ubersichtlichkeit auf das detaillierte Beschreiben der Zubereitungsweisen. Fur Ungeubte und Anfanger auf dem Gebiet sei auf die Methoden 1 und 2 verwiesen; dort sind allgemeingultige Ratschlage hierzu aufgefuhrt.

Botaniker und Zoologen konnen eine Vielzahl unterschiedlichster Pflanzen und Tiere direkt vor der „Haustur“ einsammeln, um sie im Praktikum zu demonstrieren und in vielfaltiger Art und Weise untersuchen zu lassen. Auch sind Zoologische und Botanische Garten verbreitet. Dagegen scheint es auf den ersten Blick schwierig zu sein, Mikroorganismen an ihren naturlichen Standorten ausfindig zu machen und deren Leistungen zu demonstrieren. In diesem Abschnitt des Buches stellen wir gewerbliche und kommunale Einrichtungen sowie naturliche Standorte vor, an denen Mikroorganismen „besichtigt“ werden konnen.

Cyanophycin [multi- l -arginyl-poly( l -aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae . The episomal vector systems pESC (with the galactose-inducible promoter GAL1 ) and pYEX-BX (with the copper ion-inducible promoter CUP1 ) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 ( cphA 6308 ) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA 6308 reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.

Extensive spreading of liquid manure onto agricultural fields causes eutrophication of ground and surface water and also pollution of the atmosphere due to the high ammonium nitrogen content. A poly(gamma-glutamic acid) (PGA)-producing strain of Bacillus licheniformis was isolated in this study and investigated for its ability to reduce the ammonium nitrogen by converting ammonium into biomass and PGA as depot forms of nitrogen. In batch cultivations swine manure and an optimized mineral salts medium were used for PGA production. For example the cultivation of B. licheniformis strain S2 in liquid manure, which was modified by adding of 18 g citrate and 80 g glycerol l(-1) and exhibited a carbon to nitrogen ratio of 15.5:1, led to severe reduction of the ammonium content from 2.83 to 0.1 g x l(-1) and to the production of 0.16 g PGA and 7.5 g cell dry mass l(-1) within 410 h. Approximately 28% (w/w) of the total nitrogen was converted into cellular biomass, whereas 0.1% (w/w) was used for the production of PGA. In addition, approximately 33% (w/v) of the original ammonium was lost by stripping.

Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated. One isolate was taxonomically affiliated as Pseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described. CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix. The mature form of the inducible enzyme consisted of one type of subunit with M(r) = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (beta-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression in Escherichia coli, and its nucleotide sequence was determined. The enzyme exhibited only 27-28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue. In addition, the strong inhibition of the enzyme by Pefabloc((R)) and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of beta-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism.

A 3878-bp genomic region from the cyanobacterium Synechocystis sp. strain PCC6308, amplified by inverse PCR, harbored the structural genes cphA (2625 bp) and cphB (819 bp) encoding cyanophycin synthetase and cyanophycinase, respectively. Both primary structures exhibited a high degree of similarity to the corresponding translational products from other cyanobacteria. Five regions were localized in the cyanophycin synthetase consensus sequence by their resemblance to conserved sites of ATP-dependent carboxylate-amine/thiol ligases and three substrate ligases. The functionality of cphA was proven by heterologous expression of active enzyme and synthesis of cyanophycin in Escherichia coli, which led to a maximum cyanophycin content of 26.6% (w/w) of cell dry mass. Furthermore, a modified radiometric enzyme assay for a more reliable and feasible measurement of cyanophycin synthetase activity was developed and applied to reveal the substrate specificity of the enzyme.