Interpeptide Bridge Research Articles

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus

BackgroundSynthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore β-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation.ResultsIn addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr.ConclusionSurvival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.

Terrabacter aeriphilus sp. nov., isolated from an air sample

A novel actinomycete, designated strain 5414T-18(T), was isolated from an air sample collected from the Taean region, Korea. The strain contained oxidase and grew in the presence of 7 % NaCl. A neighbour-joining tree constructed on the basis of the 16S rRNA gene sequence showed that strain 5414T-18(T) is a member of the genus Terrabacter, sharing 97.8-98.3 % 16S rRNA gene sequence similarities to type strains of species of the genus Terrabacter (98.3 % sequence similarity with Terrabacter lapilli LR-26(T)). It contained peptidoglycan containing ll-diaminopimelic acid of A3gamma type, with three glycine residues as the interpeptide bridge. Whole-cell sugars were glucose, mannose and ribose. Mycolic acids were absent. The predominant menaquinone was MK-8(H(4)). The major fatty acids (>7 % of total fatty acids) were iso-C(15 : 0), iso-C(16 : 0), C(17 : 1)omega8c and iso-C(14 : 0). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and an unidentified phosphoglycolipid. The DNA G+C content of the type strain was 73 mol%. Strain 5414T-18(T) exhibited DNA-DNA relatedness levels of 44, 43, 39, 34 and 34 % to the type strains of Terrabacter lapilli, Terrabacter aerolatus, Terrabacter terrae, Terrabacter tumescens and Terracoccus luteus, respectively. These findings suggest that strain 5414T-18(T) represents a novel species within the genus Terrabacter. The name Terrabacter aeriphilus sp. nov. is proposed for this novel species, with the type strain 5414T-18(T) (=KACC 20693(T)=DSM 18563(T)).

TRNA Involvement in Peptidoglycan Synthesis: the Work of Dieter Söll

Biosynthesis of the Peptidoglycan of Bacterial Cell Walls. VI. Incorporation of l-Threonine into Interpeptide Bridges in Micrococcus roseus (Roberts, W. S. L., Strominger, J. L., and Söll, D. (1968) J. Biol. Chem. 243, 749–756) Biosynthesis of the Peptidoglycan of Bacterial Cell Walls. VII. Incorporation of Serine and Glycine into Interpeptide Bridges in Staphylococcus epidermidis (Petit, J.-F., Strominger, J. L., and Söll, D. (1968) J. Biol. Chem. 243, 757–767) Biosynthesis of the Peptidoglycan of Bacterial Cell Walls. X. Further Study of the Glycyl Transfer Ribonucleic Acids Active in Peptidoglycan Synthesis in Staphylococcus aureus (Bumsted, R. M., Dahl, J. L., Söll, D., and Strominger, J. L. (1968) J. Biol. Chem. 243, 779–782) Dieter Söll was born in 1935. He earned his Ph.D. from Stuttgart University in 1962, after which he did a postdoctoral fellowship at the University of Wisconsin, Madison. He then joined the faculty of the University of Wisconsin and was an assistant professor there until 1967, when he joined the faculty of Yale University. Söll remains at Yale today as Sterling Professor of Molecular Biophysics and Biochemistry and Professor of Chemistry. While at the University of Wisconsin, Söll published several papers with Jack Strominger, who will be the subject of a future Journal of Biological Chemistry (JBC) Classic. In the papers, Söll and Strominger identified several tRNAs that participate in peptidoglycan formation. This was an exciting discovery as aminoacyl-tRNA was only thought to be involved in ribosome-catalyzed protein synthesis. Peptidoglycan is a polymer of sugars and amino acids that forms a homogeneous layer outside the plasma membrane of eubacteria. It serves a structural role in the bacterial cell wall, giving it shape and strength, as well as counteracting the osmotic pressure of the cytoplasm. The peptidoglycan layer is a crystal lattice formed by linear chains of N-acetylglucosamine and N-acetylmuramic acid that are connected by short (4–5-residue) amino acid chains. The nature of the bridge is to some extent genus-specific. In the first JBC Classic reprinted here, Söll, Strominger, and W. S. L. Roberts investigate the biosynthesis of peptidoglycan in Micrococcus roseus. It had previously been shown that in the formation of the Staphylococcus aureus pentaglycine peptidoglycan cross-bridge, glycine is activated, attached to tRNA, and then transferred from glycyl-tRNA to a lipid intermediate, which is utilized to form the peptidoglycan. Söll, Strominger, and Roberts isolated two threonyl-tRNAs from M. roseus R27 which catalyzed the incorporation of l-threonine onto the ϵ-amino group of lysine in the bacteria's interpeptide bridge. The threnyl-tRNAs were also able to catalyze protein biosynthesis. The second JBC Classic, which appeared back-to-back with the first paper, reports on the tRNA-dependent incorporation of serine in the interpeptide bridge of S. epidermidis. Söll, Strominger, and J.-F. Petit isolated four seryl-tRNAs involved in the transfer of serine to a lipid intermediate of peptidoglycan synthesis. However, only three of the tRNAs could be shown to participate in protein synthesis. Söll proposed several possibilities for the fourth tRNA (Ser IIA) including that “Ser IIA is a form of suppressor tRNA. That is, it would respond in protein synthesis, but to a coding triplet other than a normal serine triplet.” It was later discovered that this was indeed the bacterial selenocysteine tRNA. Twenty years later, Söll was the first to sequence the Escherichia coli selenocysteine tRNA (1Schön A. Bock A. Ott G. Sprinzl M. Söll D. The selenocysteine-inserting opal suppressor serine tRNA from E. coli is highly unusual in structure and modification.Nucleic Acids Res. 1989; 17: 7159-7165Crossref PubMed Scopus (67) Google Scholar). In the final JBC Classic, Söll, Strominger, R. M. Bumsted, and J. L. Dahl report on their isolation of three glycine acceptor tRNAs from S. aureus. All three tRNAs were active in peptidoglycan synthesis, but only two could produce polypeptides. Söll has received several awards and honors for his research, including the 1988 Alexander von Humbolt U. S. Senior Scientist Award. He is a Fellow of the American Association of the Advancement of Science and the American Academy of Microbiology and is a member of the National Academy of Sciences.

Demequina aestuarii gen. nov., sp. nov., a novel actinomycete of the suborder Micrococcineae, and reclassification of Cellulomonas fermentans Bagnara et al. 1985 as Actinotalea fermentans gen. nov., comb. nov.

An actinobacterial strain containing demethylmenaquinone DMK-9(H(4)) as the diagnostic isoprenoid quinone was isolated from a tidal flat sediment sample, from South Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain JC2054(T) represents a distinct phyletic line within the suborder Micrococcineae of the order Actinomycetales. The closest phylogenetic neighbour was Cellulomonas fermentans, with 94.7 % 16S rRNA gene sequence similarity. The novel isolate was strictly aerobic and slightly halophilic, with optimum growth occurring in 2-4 % (w/v) NaCl. Cells were non-motile, non-sporulating and rod-shaped. The peptidoglycan type was of the A-type of cross-linkage. l-ornithine was the diamino acid and d-glutamate represented the N-terminus of the interpeptide bridge. The predominant fatty acids were anteiso-branched and straight-chain fatty acids. The major polar lipids were phosphatidylinositol, diphosphatidylglycerol and an unknown phospholipid. The menaquinone composition of C. fermentans was determined to be MK-10(H(4)), MK-9(H(4)) and MK-8(H(4)) in the ratio 56 : 2 : 1. On the basis of the polyphasic evidence presented in this study, it is proposed that strain JC2054(T) should be classified as representing a novel genus and species of the suborder Micrococcineae, with the name Demequina aestuarii gen. nov., sp. nov. The type strain is JC2054(T) (=IMSNU 14027(T)=KCTC 9919(T)=JCM 12123(T)). In addition, it was clear from the phylogenetic analysis and chemotaxonomic data that C. fermentans does not belong to the genus Cellulomonas or any other recognized genera. Therefore, C. fermentans should be reclassified as representing a novel genus, for which the name Actinotalea fermentans gen. nov., comb. nov. is proposed, with strain DSM 3133(T) (=ATCC 43279(T)=CFBP 4259(T)=CIP 103003(T)=NBRC 15517(T)=JCM 9966(T)=LMG 16154(T)) as the type strain.

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA

Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to hydrolyze Gram-positive peptidoglycan (PG), while PGRP-SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP-SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP-SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP-SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.

Cell Wall-targeting Domain of Glycylglycine Endopeptidase Distinguishes among Peptidoglycan Cross-bridges

ALE-1, a homologue of lysostaphin, is a peptidoglycan hydrolase that specifically lyses Staphylococcus aureus cell walls by cleaving the pentaglycine linkage between the peptidoglycan chains. Binding of ALE-1 to S. aureus cells through its C-terminal 92 residues, known as the targeting domain, is functionally important for staphylolytic activity. The ALE-1-targeting domain belongs to the SH3b domain family, the prokaryotic counterpart of the eukaryotic SH3 domains. The 1.75 angstroms crystal structure of the targeting domain shows an all-beta fold similar to typical SH3s but with unique features. The structure reveals patches of conserved residues among orthologous targeting domains, forming surface regions that can potentially interact with some common features of the Gram-positive cell wall. ALE-1-targeting domain binding studies employing various bacterial peptidoglycans demonstrate that the length of the interpeptide bridge, as well as the amino acid composition of the peptide, confers the maximum binding of the targeting domain to the staphylococcal peptidoglycan. Truncation of the highly conserved first 9 N-terminal residues results in loss of specificity to S. aureus cell wall-targeting, suggesting that these residues confer specificity to S. aureus cell wall.

Cellulomonas bogoriensis sp. nov., an alkaliphilic cellulomonad

An alkaliphilic, slightly halotolerant, chemo-organotrophic, Gram-positive, rod-shaped bacterium, strain 69B4(T), was isolated from the sediment of the littoral zone of Lake Bogoria, Kenya. Phylogenetically, it is a member of the genus Cellulomonas, showing less than 97.5 % sequence similarity to the type strains of other Cellulomonas species. The highest level of similarity, albeit moderate, was found with respect to Cellulomonas cellasea DSM 20118(T). Chemotaxonomic properties confirm the 16S rRNA gene-based generic affiliation, i.e. a DNA G+C content of 71.5 mol%, anteiso-C(15:0) and C(16:0) as the major fatty acids, MK-9(H(4)) as the major isoprenoid quinone, a peptidoglycan containing L-ornithine as the diamino acid and D-aspartic acid in the interpeptide bridge and phosphatidylglycerol as the only identified main polar lipid. The strain is aerobic to facultatively anaerobic, being capable of growth under strictly anaerobic conditions. Optimal growth occurs between pH values 9.0 and 10.0. On the basis of its distinct phylogenetic position and metabolic properties, strain 69B4(T) represents a novel species of the genus Cellulomonas, for which the name Cellulomonas bogoriensis sp. nov. is proposed. The type strain is 69B4(T) (=DSM 16987(T)=CIP 108683(T)).

First Description of Curtobacterium spp. Isolated from Human Clinical Specimens

During a 4-year period, five strains (three of which were doubtless clinically significant) of yellow- or orange-pigmented, oxidative, slowly acid-producing coryneform bacteria were recovered from human clinical specimens in two reference laboratories or referred to them. The strains were motile, catalase positive, nitrate reductase negative, and urease negative, but strongly hydrolyzed esculin. In all reference and clinical strains described in the present study, anteisopentadecanoic (C(15:0ai)) and anteisoheptadecanoic (C(17:0ai)) acids represented more than 75% of all cellular fatty acids except in one clinical strain and in Curtobacterium pusillum, in which both the unusual omega-cyclohexyl fatty acid (identified as C(18:1omega7cis/omega9cis/omega12trans) by the Sherlock system) represented more than 50% of all cellular fatty acids. In all clinical strains, ornithine was the diamino acid of the cell wall, the interpeptide bridge consisted of ornithine, and acetyl was the acyl type of the peptidoglycan. Therefore, the five clinical strains were unambiguously identified as Curtobacterium spp. Analyses of the complete 16S rRNA genes of the five clinical strains with homologies to the established Curtobacterium species ranging from 99.2 to 100% confirmed the identifications as Curtobacterium spp. Data on the antimicrobial susceptibility pattern of curtobacteria are reported, with macrolides and rifampin showing very low MICs for all strains tested. This report is the first on the isolation of Curtobacterium strains from human clinical specimens.

Cell wall composition and decreased autolytic activity and lysostaphin susceptibility of glycopeptide-intermediate Staphylococcus aureus.

The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.

In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus.

Staphylococcus aureus peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t-RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP-MurNAc-pentapeptide, lipid I and lipid II and the staphylococcal t-RNA pool, as well as His-tagged Gly-tRNA-synthetase and His-tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II-Gly1 and lipid II-Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane-associated steps of S. aureus peptidoglycan biosynthesis.

Arthrobacter nitroguajacolicus sp. nov., a novel 4-nitroguaiacol-degrading actinobacterium.

Three bacterial isolates from soil, capable of degradation or transformation of nitroaromatic compounds and displaying a rod-coccus growth cycle, were studied by a polyphasic approach. On the basis of 16S rRNA sequence analysis and of chemotaxonomic characteristics, such as type A3alpha peptidoglycan with an interpeptide bridge Ala-Thr-Ala, the major menaquinone MK-9(H(2)) and fatty acid composition, the isolates were assigned to the genus Arthrobacter. DNA-DNA hybridization, riboprinting and phenotypic studies revealed that the three strains constitute a single species, distinct from phylogenetically neighbouring Arthrobacter aurescens and Arthrobacter ilicis. A novel species, Arthrobacter nitroguajacolicus sp. nov., with the type strain G2-1(T) (=CCM 4924(T)=DSM 15232(T)) is proposed.

Reclassification of Cellulosimicrobium variabile Bakalidou et al. 2002 as Isoptericola variabilis gen. nov., comb. nov.

As already depicted in the original publication, the type strain of the species Cellulosimicrobium variabile Bakalidou et al. 2002, DSM 10177(T), does not cluster unambiguously with the type species, Cellulosimicrobium cellulans DSM 43879(T), in phylogenetic analysis. Strain DSM 10177(T) is moderately related to the recently described species Xylanimonas cellulosilytica, Promicromonospora pachnodae and Xylanibacterium ulmi, forming a lineage that branches between C. cellulans and members of the genus Promicromonospora in most dendrograms generated on the basis of different algorithms and reference strains. The type strains of the two Cellulosimicrobium species resemble each other in morphology, composition of fatty acids, DNA G+C content, phospholipids and the presence of lysine in position 3 of the peptide subunit of peptidoglycan. However, the two strains differ from each other in cell-wall sugars and in the amino acid composition of the A4alpha-type peptidoglycan, which contains serine and aspartic acid in C. cellulans, whereas only aspartic acid is present in the interpeptide bridge of C. variabile. This type is also present in Xylanimonas cellulosilytica XIL07(T), but not in the neighbouring species P. pachnodae DSM 12657(T), which exhibits the L-lys-L-ser-D-Glu type. On the basis of distinct phylogenetic position and the amino acid composition of peptidoglycan, a novel genus and combination for C. variabile, Isoptericola variabilis gen. nov., comb. nov., is proposed.

Brachybacterium muris sp. nov., isolated from the liver of a laboratory mouse strain.

A coccoid- to ovoid-shaped, Gram-positive bacterial strain, designated C3H-21(T), was isolated from the liver of the laboratory mouse strain C3H/He and characterized by a polyphasic approach. The peptidoglycan type was variation A4gamma with meso-diaminopimelic acid as the diagnostic cell-wall diamino acid and an interpeptide bridge of D-asp-D-Glu. The isolate contained menaquinone MK-7 (88 %) as the major component of the quinone system and minor amounts of menaquinone MK-8 (9 %) and menaquinone MK-6 (3 %). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, unidentified glycolipids and unidentified phospholipids. The fatty acid profile contained predominantly anteiso-C(15 : 0) and significant amounts of iso-C(16 : 0), iso-C(14 : 0,) anteiso-C(17 : 0) and C(19 : 0). The polyamine pattern consisted of spermine and spermidine as the major compounds. Genomic fingerprints clearly distinguished strain C3H-21(T) from other Brachybacterium species. The isolate shared the highest 16S rDNA sequence similarities with members of the genus Brachybacterium, in particular Brachybacterium sacelli LMG 20345(T), Brachybacterium nesterenkovii DSM 9573(T), Brachybacterium rhamnosum LMG 19848(T), Brachybacterium alimentarium CNRZ 925(T) and Brachybacterium fresconis LMG 20336(T) (97.8-97.2 %). The results of biochemical/physiological characterization, chemotaxonomic characteristics and REP-PCR-generated fingerprints demonstrated that the isolate represents a novel species of the genus Brachybacterium, for which the name Brachybacterium muris (type strain C3H-21(T)=DSM 15460(T)=CCM 7047(T)) [corrected] is proposed.

Citricoccus muralis gen. nov., sp. nov., a novel actinobacterium isolated from a medieval wall painting.

A Gram-positive, aerobic, spherical actinobacterium, designated strain 4-0(T), was isolated from a medieval wall painting and characterized to determine its taxonomic position. The peptidoglycan of strain 4-0(T) was of type A4alpha, with lysine as the diagnostic cell wall diamino acid and an interpeptide bridge of Lys-Gly-Glu. Its quinone system contained predominantly MK-9(H2) (64%) and its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, four unknown glycolipids, two unknown phospholipids and an unknown lipid. The fatty acid profile of strain 4-0(T) was represented by significant amounts of ai-C15:0 and moderate amounts of ai-C17:0, i-C16:0 and i-C15:0 fatty acids. Spermidine was predominant in the polyamine pattern. The G+C content of the genomic DNA was 68 mol%. Comparative 16S rDNA sequence studies revealed the highest similarity values (95.4-96.1%) between strain 4-0(T) and species of the genus Micrococcus and certain species of the genus Arthrobacter, including Arthrobacter pascens DSM 20545(T), Arthrobacter ramosus DSM 20546(T), Arthrobacterprotophormiae DSM 20168(T), Arthrobacternicotianae DSM 20123(T) and Arthrobacter globiformis DSM 20124(T). Phylogenetic analyses demonstrated that strain 4-0(T) branches deeply on the Micrococcus lineage. Because it is almost equidistant phylogenetically from the genera Micrococcus and Arthrobacter and possesses significant differences in chemotaxonomic characteristics from members of these genera, it is suggested that strain 4-0(T) be classified as a novel species in a new genus; the name Citricoccus muralis gen. nov., sp. nov. is proposed. The type strain is strain 4-0(T) (= DSM 11442(T) CCM 4981(T)).

Georgenia muralis gen. nov., sp. nov., a novel actinobacterium isolated from a medieval wall painting.

Two bacterial strains, designated 1A-C(T) and 3A-1, were studied and, using these results and previously published data, taxonomically classified. Cells of the strains exhibited a rod-coccus cycle. The peptidoglycan determined for 1A-C(T) was of type A4alpha with lysine as the diagnostic cell-wall diamino acid and an interpeptide bridge of L-Lys <-- L-Glu. The menaquinone systems of the two strains contained MK-8(H4) (82-94%) and MK-7(H4) (3-11%). The polar lipid profiles consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, two unidentified phospholipids and an unidentified glycolipid. The fatty acid profiles contained predominantly ai-C15:0 and significant amounts of i-C14:0 and i-C15:1 fatty acids. Genomic fingerprints clearly distinguished strains 1A-C(T) and 3A-1 from each other. DNA-DNA relatedness between the two strains (92%) demonstrated that they are members of a single species. Analyses of the 16S rDNA sequences of strains IA-C(T) and 3A-1, which were almost identical (99.6% sequence similarity), and comparison with corresponding sequences demonstrated that they represent a novel lineage within the suborder Micrococcineae, most closely related to species of the genera Beutenbergia, Bogoriella and Cellulomonas (94.7-95.7% sequence similarity). The results demonstrate that the two strains are members of a single new genus and a single novel species. Thus, the name Georgenia muralis gen. nov., sp. nov. is proposed. The type strain is strain 1A-C(T) (= DSM 14418T = CCM 4963T). Another strain of the species is strain 3A-1 (= DSM 14419 = CCM 4964).

Structural Investigations on the Cell Surface of Erysipelothrix rhusiopathiae

The cell wall of Erysipelothrix rhusiopathiae was investigated. The corrected structure of the murein, which was published earlier, is reported here. The murein belongs to the B1delta type. It has L-alanine in position three of the peptide subunit and is crosslinked via a glycine-L-lysine-L-lysine interpeptide bridge. As expected by the complex serological situation in E. rhusiopathiae the carbohydrate moiety of the cell wall proofed to be very intricate. As accessory polymers polysaccharide(s) and teichoic acid(s) were identified. The teichoic acid(s) possess two novel polyols of which one could not be identified, the other one behaved like 6-O-methylgalactitol in gas liquid chromatography. Glucose and N-acetylgalactosamine were determined as substituents. The polysaccharide(s) is composed of mainly N-acetylfucosamine and smaller amounts of galactose, N-acetylglucosamine, and a further unidentified sugar appearing later than hexaacetylsorbitol in GLC.

Ornithinimicrobium humiphilum gen. nov., sp. nov., a novel soil actinomycete with L-ornithine in the peptidoglycan.

A Gram-positive bacterium originating from garden soil was taxonomically studied. Cells are non-motile, non-sporulating, irregular rods and cocci. The cell wall peptidoglycan contains L-ornithine as diagnostic diamino acid and an interpeptide bridge consisting of L-Orn<--L-Ala<--Gly<--D-Asp. The major menaquinone is MK-8(H4). 13-Methyl tetradecanoic acid and 14-methyl pentadecanoic acid are the predominant fatty acids. The polar lipids are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, three unknown glycolipids and three unknown phospholipids. Mycolic acids are absent. The DNA G+C composition is 70 mol%. The acyl type of the glycan chain of peptidoglycan is acetyl. Glucose is the dominating whole cell sugar; arabinose, rhamnose and xylose are present in traces. Results of 16S rDNA sequence comparisons revealed that strain HKI 0124T represents a novel lineage within the suborder Micrococcineae of the order Actinomycetales adjacent to the recently described genus Ornithinicoccus. On the basis of the clearly pronounced morphological, physiological, chemotaxonomic and phylogenetic differences between strain HKI 0124T and all members of the suborder Micrococcineae, it is proposed to assign strain HKI 0124T to a new genus and species, Ornithinimicrobium humiphilum gen. nov., sp. nov. The type and only strain is HKI 0124T (= DSM 12362T = CIP 106634T).

The fib locus in Streptococcus pneumoniae is required for peptidoglycan crosslinking and PBP-mediated beta-lactam resistance.

Penicillin resistance in pneumococci is mediated by modified penicillin-binding proteins (PBPs) that have decreased affinity to beta-lactams. In high-level penicillin-resistant transformants of the laboratory strain Streptococcus pneumoniae R6 containing various combinations of low-affinity PBPs, disruption of the fib locus results in a collapse of PBP-mediated resistance. In addition, crosslinked muropeptides are highly reduced. The fib operon consists of two genes, fibA and fibB, homologous to Staphylococcus aureus femA/B which are also required for expression of methicillin resistance in this organism. FibA and FibB belong to a family of proteins of Gram-positive bacteria involved in the formation of interpeptide bridges, thus representing interesting new targets for antimicrobial compounds for this group of pathogens.

The fib locus in Streptococcus pneumoniae is required for peptidoglycan crosslinking and PBP-mediated β-lactam resistance

Penicillin resistance in pneumococci is mediated by modified penicillin-binding proteins (PBPs) that have decreased affinity to β-lactams. In high-level penicillin-resistant transformants of the laboratory strain Streptococcus pneumoniae R6 containing various combinations of low-affinity PBPs, disruption of the fib locus results in a collapse of PBP-mediated resistance. In addition, crosslinked muropeptides are highly reduced. The fib operon consists of two genes, fibA and fibB, homologous to Staphylococcus aureus femA/ B which are also required for expression of methicillin resistance in this organism. FibA and FibB belong to a family of proteins of Gram-positive bacteria involved in the formation of interpeptide bridges, thus representing interesting new targets for antimicrobial compounds for this group of pathogens.

New LL-diaminopimelic Acid-containing Actinomycetes from Hypersaline, Heliothermal and Meromictic Antarctic Ekho Lake: Nocardioides aquaticus sp. nov. and Friedmannielly lacustris sp. nov.

Two Gram-positive, non-motile and aerobic bacteria were isolated from a water sample of the hypersaline Ekho Lake, Antarctica. The cocci or short rods grew well on oligotrophic PYGV agar of pH 7.5 and at 26 degrees C. Strains EL-17KT and EL-17AT both required thiamine and biotin, strain EL-17AT also required nicotinic acid. Carbon sources utilized by both strains were acetate, pyruvate, alpha-D-glucose, glutamate and (weakly) citrate, but succinate, malate or butyrate were utilized only by EL-17KT. Gelatin, starch and DNA were hydrolyzed, NH, was formed from peptone, and nitrate was reduced aerobically by both strains. The isolates had the same temperature tolerance for growth in the range tested (below 3 to above 33.5 degrees C) and pH range (<5.5 to >9.5) and were sensitive to chloramphenicol and penicillin G. Their cell walls contained LL-diaminopimelic acid and had a single glycine residue as interpeptide bridge. Strain EL-17AT contained glycine at position 1 of the peptide subunit (peptidoglycan type A 3gamma'). Isolates EL-17KT and EL-17AT differed in their maximum NaCl tolerance, which was 15% or 6-8%, respectively. The major fatty acid of EL-17KT was C18:1 and that of EL-17AT was ai-C15:0. The major respiratory quinones of EL-17KT and EL-17AT were MK-8(H4) and MK-9(H4), respectively. The former isolate had 69 mol% G+C, the latter had 73 mol% G+C. Comparative 16S rRNA gene sequencing revealed phylogenetic relationships of isolate EL-17KT with the genus Nocardioides, with N. pyridinolyticus and N. plantarum as the closest relatives. Phenotypic and genotypic characteristics support the description of a new species, Nocardioides aquaticus sp. nov., with EL-17KT as the type strain (= DSM 11439T). Isolate EL-17AT is related to the genus Friedmanniella, with E antarctica and E spumicola as the closest relatives. The differentiating characteristics support the description of a new species, Friedmanniella lacustris sp. nov., with EL-17AT as the type strain (= DSM 11465T).