Suborder Corynebacterineae Research Articles

Cell wall channels of Rhodococcus species: identification and characterization of the cell wall channels of Rhodococcus corynebacteroides and Rhodococcus ruber

The cell wall of Rhodococcus corynebacteroides formerly known as Nocardia corynebacteroides contains cell wall channels that are responsible for the cell wall permeability of this bacterium. Based on partial sequencing of the polypeptide subunits and a BLAST search, we identified one polypeptide of R. corynebacteroides (PorARc) and two polypeptides (PorARr and PorBRr) from the closely related bacterium Rhodococcus ruber. The corresponding genes, porARc (606 bp), porARr (702 bp), and porBRr (540 bp) are constituents of the known genome of R. corynebacteroides DSM-20151 and R. ruber DSM-43338, respectively. porARr and porBRr of R. ruber are possibly forming a common operon coding for the polypeptide subunits of the cell wall channel. The genes coding for PorARc and for PorARr and PorBRr without signal peptide were separately expressed in the porin-deficient Escherichia coli BL21DE3Omp8 strain and the proteins were purified to homogeneity. All proteins were checked for channel formation in lipid bilayers. PorARc formed channels with characteristics that were very similar to those of a previous study. The proteins PorARr and PorBRr expressed in E. coli could alone create channels in lipid bilayer membranes, despite the possibility that the two corresponding genes form a porin operon and that both subunits possibly form the cell wall channels in vivo. Based on amino acid sequence comparison of a variety of proteins forming cell wall channels in bacteria of the suborder Corynebacterineae, it seems very likely that PorARc, PorARr, and PorBRr are members of a huge family of proteins (PF09203) that form MspA-like cell wall channels.

Characterization of the Uncommon Lipid Families in Corynebacterium glutamicum by Mass Spectrometry.

This book chapter provides readers the step-by-step instruction for cell growth, lipid isolation, and lipid analysis to obtain the lipidome of Corynebacterium glutamicum (C. glutamicum) in the genus Corynebacterium, a biotechnologically important bacterium. We separate thelipid families by preparative HPLC with an analytical C-8 column, followed by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high-resolution mass measurement to define the structures of cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), α-D-mannopyranosyl-(1→4)-α-D-glucuronyl diacylglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), and acyl trehalose monomycolate (acyl-TMM) whose structures have been previously mis-assigned or not defined by mass spectrometric means. We also define the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in the cellwall. The similarity of the lipidome to that in the Mycobacterium genera is consistent with the notion that Corynebacterium and Mycobacterium are gram-positive bacteria belonging to the suborder Corynebacterineae.

Lawsonella clevelandensis as the causative agent of a breast abscess

Lawsonella clevelandensis is a Gram-stain-positive, partially acid-fast, anaerobic, being considered a new species within a new genus in the suborder Corynebacterineae. There are only a few cases reported worldwide. This is a fastidious microorganism, difficult to identify by conventional methods, leading to inappropriate treatments. The authors report a case of a 29-year-old woman with a 3-week evolution of a breast nodule. There was a family history of breast carcinoma. Samples were collected for histological and microbiological examination. Gram staining revealed Gram-positive filamentous bacilli, acid-fast-positive. The cultural examination revealed a Lawsonella clevelandensis that was confirmed by molecular methods. At the last follow up, the evolution was favorable; the abscess was resolved, with no evidence of recurrence. To our knowledge the present case was the first reported in Europe.

Extracytoplasmic function sigma factor σD confers resistance to environmental stress by enhancing mycolate synthesis and modifying peptidoglycan structures in Corynebacterium glutamicum.

Mycolates are α-branched, β-hydroxylated, long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. They form an outer membrane, which functions as a permeability barrier and confers pathogenic mycobacteria to resistance to antibiotics. Although the mycolate biosynthetic pathway has been intensively studied, knowledge of transcriptional regulation of genes involved in this pathway is limited. Here, we report that the extracytoplasmic function sigma factor σD is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σD -binding regions in the genome, establishing a consensus promoter sequence for σD recognition. The σD regulon comprised acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase and mycolyltransferases; they were involved in mycolate synthesis. Indeed, deletion or overexpression of sigD encoding σD modified the extractable mycolate amount. Immediately downstream of sigD, rsdA encoded anti-σD and was under the control of a σD -dependent promoter. Another σD regulon member, l,d-transpeptidase, conferred lysozyme resistance. Thus, σD modifies peptidoglycan cross-linking and enhances mycolate synthesis to provide resistance to environmental stress.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

Reclassification of Amycolicicoccus subflavus as Hoyosella subflava comb. nov. and emended descriptions of the genus Hoyosella and Hoyosella altamirensis.

16S rRNA gene sequences of two type strains belonging to different genera within the suborder Corynebacterineae, namely Hoyosella altamirensis and Amycolicicoccus subflavus, show a similarity of 99.8 %. Therefore, in order to clarify their taxonomic relationship, a polyphasic recharacterization under the same conditions was carried out. The peptidoglycan of H. altamirensis NBRC 109631T and A. subflavus NBRC 109087T was of A1γ type with meso-diaminopimelic acid as their diagnostic diamino acid. Both strains contained MK-8 as the only detected menaquinone, C16 : 0 and C18 : 1ω9c as the major fatty acids, and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as the principal polar lipids. The coincidences of these chemotaxonomic features suggested that H. altamirensis and A. subflavus should be assigned to the same genus. Meanwhile, the average nucleotide identity value between both strains and the results of physiological and biochemical tests indicated that H. altamirensis and A. subflavus should be affiliated to different species. Therefore, according to Rules 38 and 41a of the Bacteriological Code, it is proposed that Amycolicicoccus subflavus Wang et al. 2010 be reclassified as Hoyosella subflava comb. nov. (type strain DQS3-9A1T=CGMCC 4.3532T=DSM 45089T=JCM 17490T=NBRC 109087T) and the descriptions of the genus HoyosellaJurado et al. 2009 and Hoyosella altamirensisJurado et al. 2009 are emended accordingly.

Lawsonella clevelandensis gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from human abscesses.

Gram-stain-positive, partially acid-fast, non-spore-forming, anaerobic, catalase-positive, pleomorphic bacteria were isolated from human abscesses. Strains X1036T, X1698 and NML 120705, were recovered from a spinal abscess, a peritoneal abscess and a breast abscess respectively. A phylogenetic analysis of the 16S rRNA gene sequences showed that the strains shared 100 % similarity, and the nearest phylogenetic neighbour was Dietzia timorensis DSM 45568T (95%). Chemotaxonomic characteristics of the strains were consistent with those described for members of the suborder Corynebacterineae. Mycolic acids were detected using HPLC and one-dimensional TLC; whole-cell hydrolysates yielded meso-diaminopimelic acid with arabinose and galactose as the predominant sugars; the muramic acid acyl type was acetylated; the major menaquinone was MK-9 (96.3%); polar lipids detected were phosphatidylglycerol, phosphatidylinositol and an unknown glycophospholipid. Cellular fatty acids were hexadecanoic acid (C16 : 0), octadecenoic acid (C18 : 1ω9c) and decanoic acid (C10 : 0). Tuberculostearic acid was not detected. Based on the results of this polyphasic study, we conclude that these strains represent a novel genus and species within the suborder Corynebacterineae for which we propose the name Lawsonella clevelandensis gen. nov., sp. nov., with the type strain X1036T (=DSM 45743T=CCUG 66657T).

Genome-wide bioinformatics analysis of steroid metabolism-associated genes in Nocardioides simplex VKM Ac-2033D

Actinobacteria comprise diverse groups of bacteria capable of full degradation, or modification of different steroid compounds. Steroid catabolism has been characterized best for the representatives of suborder Corynebacterineae, such as Mycobacteria, Rhodococcus and Gordonia, with high content of mycolic acids in the cell envelope, while it is poorly understood for other steroid-transforming actinobacteria, such as representatives of Nocardioides genus belonging to suborder Propionibacterineae. Nocardioides simplex VKM Ac-2033D is an important biotechnological strain which is known for its ability to introduce ∆(1)-double bond in various 1(2)-saturated 3-ketosteroids, and perform convertion of 3β-hydroxy-5-ene steroids to 3-oxo-4-ene steroids, hydrolysis of acetylated steroids, reduction of carbonyl groups at C-17 and C-20 of androstanes and pregnanes, respectively. The strain is also capable of utilizing cholesterol and phytosterol as carbon and energy sources. In this study, a comprehensive bioinformatics genome-wide screening was carried out to predict genes related to steroid metabolism in this organism, their clustering and possible regulation. The predicted operon structure and number of candidate gene copies paralogs have been estimated. Binding sites of steroid catabolism regulators KstR and KstR2 specified for N. simplex VKM Ac-2033D have been calculated de novo. Most of the candidate genes grouped within three main clusters, one of the predicted clusters having no analogs in other actinobacteria studied so far. The results offer a base for further functional studies, expand the understanding of steroid catabolism by actinobacteria, and will contribute to modifying of metabolic pathways in order to generate effective biocatalysts capable of producing valuable bioactive steroids.

Bioorthogonal Chemical Reporters for Selective In Situ Probing of Mycomembrane Components in Mycobacteria

AbstractThe global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non‐covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria‐specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non‐covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.

Bioorthogonal Chemical Reporters for Selective In Situ Probing of Mycomembrane Components in Mycobacteria.

The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.

Characterization of Mycolic Acids in Total Fatty Acid Methyl Ester Fractions from Mycobacterium Species by High Resolution MALDI-TOFMS.

Mycolic acids (MAs) are characteristic components of bacteria in the suborder Corynebacterineae, such as Mycobacterium. MAs are categorized into subclasses based on their functional bases (cyclopropane ring, methoxy, keto, and epoxy group). Since MAs have heterogeneity among bacterial species, analyzing of MAs are required in the chemotaxonomic field. However, their structural analysis is not easy because of their long carbon-chain lengths and several functional groups. In this study, total fatty acid (FA) methyl ester (ME) fraction of M. tuberculosis H37Rv was analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS). The distributions of carbon-chain length and their relative peak intensities were confirmed with those obtained by analysis of each subclass fraction which was separated from total FA ME fraction using thin-layer chromatography (TLC). The observed major peaks were reliably assigned as MAs owing to the high mass accuracy (error<3 ppm). The types of MA subclasses, their distributions of carbon-chain lengths, their relative peak intensities, and the ratio of even- and odd-numbered carbon-chain MAs for the total FA ME fraction were consistent with those of MA subclass fractions. To visualize whole MAs, contour maps of relative peak intensities for whole MAs were created. The contour maps indicated the MA subclasses and their distributions of carbon-chains with relative peak intensities at a glance. Our proposed method allows simple characterization in a short time and thus enables the analysis of large numbers of samples, and it would contribute to the chemotaxonomy.

Cell division in Corynebacterineae.

Bacterial cells must coordinate a number of events during the cell cycle. Spatio-temporal regulation of bacterial cytokinesis is indispensable for the production of viable, genetically identical offspring. In many rod-shaped bacteria, precise midcell assembly of the division machinery relies on inhibitory systems such as Min and Noc. In rod-shaped Actinobacteria, for example Corynebacterium glutamicum and Mycobacterium tuberculosis, the divisome assembles in the proximity of the midcell region, however more spatial flexibility is observed compared to Escherichia coli and Bacillus subtilis. Actinobacteria represent a group of bacteria that spatially regulate cytokinesis in the absence of recognizable Min and Noc homologs. The key cell division steps in E. coli and B. subtilis have been subject to intensive study and are well-understood. In comparison, only a minimal set of positive and negative regulators of cytokinesis are known in Actinobacteria. Nonetheless, the timing of cytokinesis and the placement of the division septum is coordinated with growth as well as initiation of chromosome replication and segregation. We summarize here the current knowledge on cytokinesis and division site selection in the Actinobacteria suborder Corynebacterineae.

A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

BackgroundArginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, “classical” ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene.ResultsArginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families.ConclusionsThe N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising amongst others the genera Corynebacterium, Mycobacterium, and Nocardia. Therefore, the name C-NAGS (Corynebacterineae-type NAGS) is proposed for this new family.

Mycolic acids – potential biomarkers of opportunistic infections caused by bacteria of the suborder Corynebacterineae

Mycolic acids are one of the basic elements of the cell wall structure of bacteria belonging to the suborder Corynebacterineae, constituting from 20% to 40% of dry weight. Additionally, they show high structural diversity within each family and species. Nowadays, profiles of mycolic acids are widely described for the genus Mycobacterium, the causative agent of tuberculosis. However, the suborder Corynebacterineae also includes many representatives of opportunistic human pathogens, e.g. Dietzia, Gordonia, Nocardia and Rhodococcus. Currently, an increased infection risk caused by this group of microorganisms especially in immunocompromised patients has been observed. Better knowledge of mycolic acid profiles for Corynebacterineae may allow identification of mycolic acids as diagnostic markers in the detection of opportunistic bacterial infections. Modern techniques of chemical analysis, including mass spectrometry, may enable the development of new chemotaxonomic methods for the detection and differentiation of bacteria within the suborder Corynebacterineae.

Identification of a Novel Gene Product That Promotes Survival of Mycobacterium smegmatis in Macrophages

BackgroundBacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs.Methodology/Principal FindingsWe have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate.Conclusions/Significance MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.

Decaprenylphosphoryl-β-D-Ribose 2′-Epimerase, the Target of Benzothiazinones and Dinitrobenzamides, Is an Essential Enzyme in Mycobacterium smegmatis

BackgroundThe unique cell wall of bacteria of the suborder Corynebacterineae is essential for the growth and survival of significant human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Drug resistance in mycobacteria is an increasingly common development, making identification of new antimicrobials a priority. Recent studies have revealed potent anti-mycobacterial compounds, the benzothiazinones and dinitrobenzamides, active against DprE1, a subunit of decaprenylphosphoribose 2′ epimerase which forms decaprenylphosphoryl arabinose, the arabinose donor for mycobacterial cell wall biosynthesis. Despite the exploitation of Mycobacterium smegmatis in the identification of DprE1 as the target of these new antimicrobials and its use in the exploration of mechanisms of resistance, the essentiality of DprE1 in this species has never been examined. Indeed, direct experimental evidence of the essentiality of DprE1 has not been obtained in any species of mycobacterium.Methodology/Principal FindingsIn this study we constructed a conditional gene knockout strain targeting the ortholog of dprE1 in M. smegmatis, MSMEG_6382. Disruption of the chromosomal copy of MSMEG_6382 was only possible in the presence of a plasmid-encoded copy of MSMEG_6382. Curing of this “rescue” plasmid from the bacterial population resulted in a cessation of growth, demonstrating gene essentiality.Conclusions/SignificanceThis study provides the first direct experimental evidence for the essentiality of DprE1 in mycobacteria. The essentiality of DprE1 in M. smegmatis, combined with its conservation in all sequenced mycobacterial genomes, suggests that decaprenylphosphoryl arabinose synthesis is essential in all mycobacteria. Our findings indicate a lack of redundancy in decaprenylphosphoryl arabinose synthesis in M. smegmatis, despite the relatively large coding capacity of this species, and suggest that no alternative arabinose donors for cell wall biosynthesis exist. Overall, this study further validates DprE1 as a promising target for new anti-mycobacterial drugs.

Transcriptional regulation of gene expression inCorynebacterium glutamicum: the role of global, master and local regulators in the modular and hierarchical gene regulatory network

The genus Corynebacterium belongs to the taxonomic class Actinobacteria representing the high G+C branch of gram-positive bacteria. Among the most prominent members of this genus is Corynebacterium glutamicum, which is used by the biotechnological industry for the fermentative production of l-amino acids. Because of its industrial importance, C. glutamicum is one of the best-studied gram-positive bacteria and an emerging model for the suborder Corynebacterineae. Over the past years, the transcriptional regulation of gene expression was intensively studied in C. glutamicum to gain insights into the regulatory gene composition and the architecture of the transcriptional regulatory network. Therefore, we have achieved considerable improvements in understanding the regulatory roles of the various transcription regulators and the topology of the gene-regulatory network. This review summarizes the current knowledge of the regulatory gene repertoire and the reconstruction of the transcriptional regulatory network. We describe the functional classification of the detected transcription regulators and their interactions in the gene-regulatory network. Moreover, we provide an overview of the molecular features of transcription regulators and their regulons to integrate these data into the gene-regulatory network and to unravel the flow of information from the environment toward the gene level.

Tomitella biformata gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from a permafrost ice wedge

Gram-reaction-positive, aerobic, non-spore-forming, irregular rod-shaped bacteria, designated AHU1821(T) and AHU1820, were isolated from an ice wedge in the Fox permafrost tunnel, Alaska. The strains were psychrophilic, growing at -5 to 27°C. Phylogenetic analysis of the 16S rRNA and gyrB gene sequences indicated that the ice-wedge isolates formed a clade distinct from other mycolic-acid-containing bacteria within the suborder Corynebacterineae. The cell wall of strains AHU1821(T) and AHU1820 contained meso-diaminopimelic acid, arabinose and galactose, indicating chemotype IV. The muramic acids in the peptidoglycan were glycolated. The predominant menaquinone was MK-9(H(2)). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unidentified glycolipid. The major fatty acids were hexadecenoic acid (C(16 : 1)), hexadecanoic acid (C(16 : 0)), octadecenoic acid (C(18 : 1)) and tetradecanoic acid (C(14 : 0)). Tuberculostearic acid was present in relatively small amounts (1 %). Strains AHU1821(T) and AHU1820 contained mycolic acids with 42-52 carbons. The DNA G+C content of the two strains was 69.3-71.6 mol% (T(m)). 16S rRNA, rpoB and recA gene sequences were identical between strains AHU1821(T) and AHU1820 and those of the gyrB gene showed 99.9 % similarity. Based on phylogenetic and phenotypic evidence, strains AHU1821(T) and AHU1820 represent a single novel species of a novel genus, for which the name Tomitella biformata gen. nov., sp. nov. is proposed. The type strain of Tomitella biformata is AHU1821(T) (=DSM 45403(T) =NBRC 106253(T)).

The transcriptional regulatory repertoire of Corynebacterium glutamicum: Reconstruction of the network controlling pathways involved in lysine and glutamate production

Corynebacterium glutamicum is one of the best studied organisms of the high G + C branch of Gram-positive bacteria and an emerging model system for the suborder Corynebacterineae. To gain insights into the regulatory gene composition and architecture of the transcriptional regulatory network of C. glutamicum, components of the transcriptional regulatory repertoire were intensively studied by many scientific groups in recent years. In this mini-review, we summarize the present knowledge about the deduced transcriptional regulatory repertoire of C. glutamicum and the current status of transcriptional regulatory network reconstruction with regard to the genome-wide detection of transcriptional regulations, coregulatory interactions and hierarchical cross-regulations. Moreover, we provide an overview of those regulators and their transcriptional regulations controlling genes involved in the conversion of the carbon sources glucose, fructose and sucrose into the industrially relevant products l-lysine and l-glutamate. This data will contribute to our understanding of l-lysine and l-glutamate production by C. glutamicum from the perspective of systems biology and may provide the basis for computational modeling of the respective biotechnologically important metabolic pathways.

Phylogenetic studies of Nocardia species based on gyrB gene analyses

Phylogenetic analyses of 56 type species of Nocardia were conducted using the partial nucleotide sequences of the gyrase B-encoding gene (gyrB). The interspecies similarities of the gyrB gene for the 56 type species were 82.4-99.9 %, which corresponded to 270-2 nt differences in the partial gene sequences of approximately 1200 nt. In comparison with phylogenetic relationships, gyrB gene sequence information was generally consistent with that of 16S rRNA gene sequences with minor exceptions. However, the degree of divergence of the gyrB gene sequences was approximately 3.6 times greater than those of the 16S rRNA gene, suggesting a higher discriminative power of gyrB sequence information compared with 16S rRNA gene sequences for Nocardia species. The Nocardia type species were clustered based on gyrB sequence similarity values of 93.5 % and above. Among the 56 type species, 38 were distributed in 13 clusters, each comprising 2 to 7 species. The remaining 18 species were classified into an independent cluster, in which the similarity between each species and the other 55 Nocardia species was less than 93.5 %. Among the eight mycolic acid-containing actinomycete genera in the suborder Corynebacterineae, Nocardia was clearly differentiated from the other genera, such as Rhodococcus, by gyrB gene analyses (similarity values of gyrB sequences for Nocardia and Rhodococcus were 75-85 %), indicating that the gyrB gene is a useful alternative to the 16S rRNA gene for the determination of phylogenetic relationships between the genus Nocardia and the seven other actinomycete genera.