Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteria

A phylogenetic tree constructed by 5S rRNA analysis is composed of three major branches in eubacteria: high G + C Gram+, low G + C Gram+ and Gram- [Hori and Osawa, Mol. Biol. Evol. 4 (1987) 445-472]. We have shown that the characteristic dnaA region is common among Escherichia coli (Gram-), Pseudomonas putida (Gram-), and Bacillus subtilis (low G + C Gram+). We have now determined the structure of the dnaA region of Micrococcus luteus, as a representative of the last branch, high G + C Gram+. The dnaA gene and at least three other genes, rnpA, rpmH and dnaN were found to be conserved in M. luteus. Large nontranslatable regions were found flanking the dnaA gene. The upstream region is conserved in the four bacteria so far examined. On the other hand, the downstream region is conserved only in Gram+ bacteria, M. luteus and B. subtilis. The consensus sequence of the DnaA box in M. luteus seems to be TTGTCCACA, in contrast to TTATCCACA of other bacteria. These results confirm our hypothesis that the dnaA region is the replication origin of the ancestral bacteria and that the essential feature of the DnaA protein and DnaA-box combination is conserved in eubacteria.     

腾格里沙漠东南缘可培养微生物群落数量与结构特征

以腾格里沙漠东南缘沙漠化土壤为研究对象,研究了不同沙漠化修复程度土壤结皮及结皮下微生物分布特征及多样性。结果表明:研究区域可培养细菌数量随沙漠化生态修复进程呈升高趋势,随采样深度呈下降趋势。数量以节杆菌属和芽孢杆菌属为主,其含量随沙漠化修复程度分别呈降低与升高趋势。修复过程中可培养土壤微生物数量与土壤碳、氮含量呈极显著正相关关系,与pH值呈极显著负相关关系,说明微生物数量与沙漠化土壤改良程度密切相关。通过16S rDNA基因测序及构建系统发育树,研究区域可培养细菌归类为18个属,分属于6个系统发育组:高G+C革兰氏阳性类群、低G+C革兰氏阳性类群、α-变形菌、β-变形菌、γ-变形菌和CFB类群,序列比对显示菌株功能多样。     

Structure of the dnaA and DnaA-box region in the Mycoplasma capricolum chromosome: conservation and variations in the course of evolution.

We have previously shown that the dnaA gene and the DnaA-box region were conserved in bacteria representative of all three major branches of the eubacterial phylogenic tree: high G + C Gram+, low-G + C Gram+ and Gram-. In the present work, we determined the structure of the dnaA region of Mycoplasma capricolum and found that the dnaA gene and at least two other genes, rpmH and dnaN, were conserved in this bacterium. An unusually high level of amino acid (aa) substitutions was observed in M. capricolum DnaA. It was the case even in those aa which were well conserved in other bacterial species. The nontranslatable region upstream from the dnaA gene was also conserved in this bacterium, as it was universally found in both Gram+ and Gram- bacteria. An additional nontranslatable region downstream from the dnaA gene, which is common to Gram+ bacteria, was also found in M. capricolum, consistent with the proposal that M. capricolum is Gram+ in origin. These regions were rich in A + T and contained ten DnaA-box-like sequences (9-mers that differ from TTATCCACA by one or two bases).     

The transcriptional regulator Rok binds A+T-rich DNA and is involved in repression of a mobile genetic element in Bacillus subtilis

The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.     

Enterococcus adhesin PrgB facilitates type IV secretion by condensation of extracellular DNA

Conjugative type IV secretion systems (T4SSs) are multi‐protein complexes in Gram‐negative and Gram‐positive (G+) bacteria, responsible for spreading antibiotic resistances and virulence factors among different species. Compared to Gram‐negative bacteria, which establish close contacts for conjugative transfer via sex pili, G+ T4SSs are suggested to employ surface adhesins instead. One example is pCF10, an enterococcal conjugative sex‐pheromone responsive plasmid with a narrow host range, thus disseminating genetic information only among closely related species. This MicroCommentary is dedicated to the crystal structure of the pCF10‐encoded adhesion domain of PrgB presented by Schmitt et al. The authors show in their work that this adhesion domain is responsible for biofilm formation, tight binding and condensation of extracellular DNA (eDNA) and conjugative transfer of pCF10. A sophisticated two‐step mechanism for highly efficient conjugative transfer is postulated, including the formation of PrgB‐mediated long‐range intercellular contacts by binding and establishment of shorter‐range contacts via condensation of eDNA. PrgB binding to lipoteichoic acid on the recipient cell surface stabilizes junctions between the mating partners. The major findings by Schmitt et al. will be brought into a broader context and potential medical applications targeting eDNA as essential component in biofilm formation and conjugation will be discussed.     

Evolutionary relationships of <Emphasis Type="Italic">Fusobacterium nucleatum</Emphasis> based on phylogenetic analysis and comparative genomics

Background  

The phylogenetic position and evolutionary relationships of Fusobacteria remain uncertain. Especially intriguing is their relatedness to low G+C Gram positive bacteria (Firmicutes) by ribosomal molecular phylogenies, but their possession of a typical gram negative outer membrane. Taking advantage of the recent completion of the Fusobacterium nucleatum genome sequence we have examined the evolutionary relationships of Fusobacterium genes by phylogenetic analysis and comparative genomics tools.     

Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study, Corynebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glutamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

The Limulus Lysate Endotoxin Assay as a Test of Microbial Quality of Ground Beef

The Limulus lysate test (LLT) for endotoxin assay has been found to be an excellent, simple and rapid test of microbial quality of refrigerated ground beef. In fresh ground beef held at 5°C for 7–12 d, LLT titres increased from 10²–10⁵ and correlated very highly with extract-release volume (ERV) data and total viable Gram negative counts at both 5° and 30°C. The LLT was negative for fresh beef containing low numbers of bacteria and on aged beef in the absence of increasing numbers of Gram negative bacteria. Of 14 Gram negative meat isolates, all gave a positive LLT while none of eight miscellaneous Gram positive bacteria did. The use of this test provides objective information on the microbial quality of fresh refrigerated ground meats in 1 h. Based upon this study, it is suggested that a 0·1 ml inoculum from a 10³ dilution of good quality ground beef should produce a negative lysate test and thus serve as an additional rapid screening test of meat microbial quality.     

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium Salmonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.     

Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens

This study investigated the antibacterial activities of sophoraflavanone G from Sophora flavescens in combination with two antimicrobial agents against oral bacteria. The combined effect of sophoraflavanone G and the antimicrobial agents was evaluated using the checkerboard method to obtain a fractional inhibitory concentration (FIC) index. The sophoraflavanone G+ampicillin (AM) combination was found to have a synergistic effect against S. mutans, S. sanguinis, S. sobrinus, S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis, whereas the sophoraflavanone G+gentamicin (GM) combination had a synergistic effect against S. sanguinis, S. criceti, S. anginosus, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Neither combination exhibited any antagonistic interactions (FIC index >4). In particular, the MICs/MBCs for all the bacteria were reduced to one-half - one-sixteenth as a result of the drug combinations. A synergistic interaction was also confirmed by time-kill studies for nine bacteria where the checkerboard suggested synergy. Thus, a strong bactericidal effect was exerted through the drug combinations, plus in vitro data suggested that sophoraflavanone G combined with other antibiotics may be microbiologically beneficial rather than antagonistic.     

Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.     

Chemical compositions and antimicrobial activities of four different Anatolian propolis samples

Propolis means a gum that is gathered by bees from various plants. It is known for its biological properties, having antibacterial, antifungal and healing properties. The aims of this study were to evaluate the antimicrobial activity of four different Anatolian propolis samples on different groups of microorganisms including some oral pathogens and comparison between their chemical compositions. Ethanol extracts of propolis (EEP) were prepared from four different Anatolian propolis samples and examined whether EEP inhibit the growth of the test microorganisms or not. For the antimicrobial activity assays, minimum inhibitory concentrations (MIC) were determined by using macrodilution method. The MIC values of the most effective propolis (TB) were 2 microg/ml for Streptococcus sobrinus and Enterococcus faecalis, 4 microg/ml for Micrococcus luteus, Candida albicans and C. krusei, 8 microg/ml for Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis and Enterobacter aerogenes, 16 microg/ml for Escherichia coli and C. tropicalis and 32 microg/ml for Salmonella typhimurium and Pseudomonas aeruginosa. The chemical compositions of EEP's were determined by high-temperature high-resolution gas chromatography coupled to mass spectrometry. The main compounds of four Anatolian propolis samples were flavonoids such as pinocembrin, pinostropin, isalpinin, pinobanksin, quercetin, naringenin, galangine and chrysin. Although propolis samples were collected from different regions of Anatolia all showed significant antimicrobial activity against the Gram positive bacteria and yeasts. Propolis can prevent dental caries since it demonstrated significant antimicrobial activity against the microorganisms such as Streptococcus mutans, Streptococcus sobrinus and C. albicans, which involves in oral diseases.     

Diversity,Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.     

Structure and functional analysis of the microbial community in an aerobic: anaerobic sequencing batch reactor (SBR) with no phosphorus removal

The bacterial community of an aerobic:anaerobic non-P removing SBR biomass fed a mixture of acetate and glucose was analysed using several 16S rRNA based methods. Populations responsible for anaerobic glucose and acetate assimilation were determined with fluorescent in situ hybridization (FISH) in combination with microautoradiography (FISH/MAR). At 'steady state' this community consisted of alpha-Proteobacteria (26%) and gamma-Proteobacteria (14%), mainly appearing as large cocci in tetrads (i.e. typical 'G-Bacteria'). Large numbers of low G+C bacteria (22%), and high G+C Gram-positive bacteria (29%) seen as small cocci in clusters or in sheets were also detected after FISH. DGGE fingerprinting of PCR amplified 16S rDNA fragments and subsequent cloning and sequencing of several of the major bands led to the identification of some of these populations. They included an organism 98% similar in its 16S rRNA sequence to Micropruina glycogenica, and ca. 76% of the high G+C bacteria responded to a probe MIC 184, designed against it. The rest responded to the KSB 531 probe designed against a high G+C clone sequence, sbr-gs28 reported in other similar systems. FISH analyses showed that both these high G+C populations were almost totally dominated by small clustered cocci. Only ca. 2% of cells were beta-Proteobacteria. None of the alpha- and gamma-Proteobacterial 'G-bacteria' responded to FISH probes designed for the 'G-Bacteria' Amaricoccus spp. or Defluvicoccus vanus. FISH/MAR revealed that not all the alpha-Proteobacterial 'G-Bacteria' could take up acetate or glucose anaerobically. Almost all of the gamma-Proteobacterial 'G-Bacteria' assimilated acetate anaerobically but not glucose, the low G+C clustered cocci only took up glucose, whereas the high G+C bacteria including M. glycogenica and the sbr-gs28 clone assimilated both acetate and glucose. All bacteria other than the low G+C small cocci and a few of the alpha-Proteobacteria accumulated PHB. The low G+C bacteria showing anaerobic glucose assimilation ability were considered responsible for the lactic acid produced anaerobically by this SBR biomass, and M. glycogenica for its high glycogen content.     

Molecular fingerprinting of dairy microbial ecosystems by use of temporal temperature and denaturing gradient gel electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of approximately 150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.     

Biological activity of 4-hydroxy-5-formylbenzoic acid derivatives. II. Esters and Schiff bases with antimicrobial activity

A number of hitherto undescribed 4-hydroxy-5-formylbenzoic acid derivatives (A), have been prepared and characterized. (formula; see text) Esters (X = CH3) and Schiff's bases (Z = N-aryl) were prepared by conventional methods and were obtained in satisfactory yield and in a good state of purity. The prepared compounds have been tested for "in vitro" activity against Gram+ bacteria (S.epidermidis, B.subtilis, B.anthracis, M.paratuberculosis), Gram- bacteria (P.aeruginosa, S.typhi murium, E.coli Bb, S.typhi O, S.typhi infantis, S.paratyphi A, S.paratyphi B) and fungi (C.albicans, A.niger, S.cerevisiae) by agar-diffusion method (Kirby-Bauer modified). The prepared compounds, generally, showed inhibitory activity against Gram+ bacteria. Esters (A: X = CH3) showed antibacteric and antimycotic activity. The greatest activity was observed in the methyl ester (XV) of 4-hydroxy-5-formylbenzoic acid (I).     

DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract

A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.     

Polyamines in representative taxa of coryneform and other bacteria

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.