<Emphasis Type="Italic">Streptomyces tunisialbus</Emphasis> sp. nov., a novel <Emphasis Type="Italic">Streptomyces</Emphasis> species with antimicrobial activity

A novel actinomycete strain designated S2^T was isolated from Tunisian rhizosphere soil of Lavandula officinalis. This isolate exhibited broad spectrum antibacterial activity against several Gram-positive and Gram-negative bacteria and also antifungal activity against yeast and filamentous fungi. The isolate S2^T presents morphological and chemotaxonomic characteristics typical of the members of the genus Streptomyces. Whole cell hydrolysates of S2^T were found to contain LL-diaminopimelic acid. The major fatty acids were identified as C16:0, anteiso-C15:0 and iso-C16:0 whereas the predominant menaquinones were found to be MK-9(H6) and MK-9(H8). The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and three unidentified compounds. The G+C content of the genomic DNA was determined to be 71.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S2^T belongs to the genus Streptomyces and is closely related to Streptomyces netropsis DSM 40259^T with 99.86% sequence similarity. Multi-locus sequence analysis (MLSA) based on four house-keeping gene alleles (gyrB, recA, trpB, rpoB) showed that isolate S2^T is closely related to S. netropsis, with an MLSA distance greater than 0.007. The DNA–DNA relatedness between strain S2^T and its near phylogenetic neighbour was 63.6 ± 2.3%, which is lower than the 70% threshold value for delineation of genomic prokaryotic species. This isolate was also distinguished from the type strain S. netropsis DSM 40259^T, using a combination of morphological and physiological features. Based on its phenotypic and molecular properties, strain S2^T is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces tunisialbus sp. nov. is proposed. The type strain is S2^T (= JCM 32165^T = DSM 105760^T).     

<Emphasis Type="Italic">Trueperella pyogenes</Emphasis> isolated from a brain abscess of an adult roebuck (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S–23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.     

<Emphasis Type="Italic">Kribbella podocarpi</Emphasis> sp. nov., isolated from the leaves of a yellowwood tree (<Emphasis Type="Italic">Podocarpus latifolius</Emphasis>)

An endophytic actinobacterial strain was isolated from a yellowwood tree growing on the slope of Devil’s Peak, Cape Town, South Africa. Analysis of the 16S rRNA gene showed that the strain belongs to the genus Kribbella. Phylogenetic analyses using the 16S rRNA gene and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes showed that strain YPL1^T is closely related to the type strains of Kribbella karoonensis and Kribbella shirazensis. DDH experiments showed that strain YPL1^T is a distinct genomic species from its close phylogenetic relative, K. karoonensis Q41^T. Physiological comparisons further showed that strain YPL1^T is phenotypically distinct from the type strains of Kribbella jejuensis, Kribbella aluminosa, K. karoonensis, K. shirazensis and Kribbella swartbergensis. Strain YPL1^T is thus presented as the type strain of a novel species, for which the name Kribbella podocarpi sp. nov. (= DSM 29424^T = NRRL B-65063^T), is proposed.     

<Emphasis Type="Italic">Haematospirillum</Emphasis> and insect <Emphasis Type="Italic">Wolbachia</Emphasis> DNA in avian blood

In this study, blood samples of 259 Acrocephalus sp. warblers were molecularly analysed for Anaplasmataceae and Rhodospirillaceae based on PCR amplification of 16S rRNA gene fragments. One bird blood sample (from Reed Warbler, Acrocephalus scirpaceus) yielded a sequence with 99.8% identity to Haematospirillum jordaniae. This is the first molecular evidence for the occurrence of this species in the blood of any vertebrate other than human. Another bird blood sample (from Marsh Warbler: Acrocephalus palustris) yielded a Wolbachia sequence, closely related to a moth endosymbiont with 99.8% identity. A nematode origin of Wolbachia DNA detected here in avian blood can be excluded, because results of phylogenetic analysis showed its closest alignment with insect wolbachiae. This is the first finding of insect Wolbachia DNA in the circulatory system of birds, which can be explained either by the inoculation of wolbachiae by blood-sucking vectors, or passing of Wolbachia DNA from the gut into the blood of this insectivorous bird species.     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Molecular diversity and genetic relationships in <Emphasis Type="Italic">Secale</Emphasis>

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.     

<Emphasis Type="Italic">Sphingorhabdus buctiana</Emphasis> sp. nov., isolated from fresh water,and reclassification of <Emphasis Type="Italic">Sphingopyxis contaminans</Emphasis> as <Emphasis Type="Italic">Sphingorhabdus contaminans</Emphasis> comb. nov.

A Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, designated T5^T, was isolated from the Chishui River in Maotai town, Guizhou Province, Southwest of China. Strain T5^T was found to grow optimally at pH 9.0 and 25 °C. The 16S rRNA gene sequence analysis indicated that strain T5^T belongs to the family Sphingomonadaceae within the phylum Proteobacteria; the strain T5^T clustered with the type strains of Sphingopyxis contaminans, Sphingorhabdus wooponensis and Sphingorhabdus rigui, with which it exhibits 16S rRNA gene sequence similarity values of 96.2–96.9%. The DNA G+C content was 58.5 mol%. The major respiratory quinone was Q-10 and the major polar lipid was phosphatidylethanolamine. The major polyamine was homospermidine and the major fatty acids were C_18:1 ω7c (37.5%) and C_16:1 ω7c (30.1%). On the basis of phylogenetic, phenotypic and genetic data, strain T5^T represents a novel species of the genus Sphingorhabdus, for which the name Sphingorhabdus buctiana sp. nov. is proposed. The type strain is T5^T (= CGMCC 1.12929^T = JCM 30114^T). It is also proposed that Sphingopyxis contaminans should be reclassified as a member of the genus Sphingorhabdus.     

Genetic diversity of the natural strains of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

The method of RAPD-PCR and comparative analysis of the PCR fingerprinting profiles similarity was used to characterize interspecific diversity of natural isolates of the lactic acid bacteria Streptococcus thermophilus. The strain genetic diversity was demonstrated using three primer variants, designed for different bacterial genome regions. The resolution of RAPD-PCR technique with different primers for identification at the species level and for certification at the strain level, was examined relative to the commercially important cultures of S. thermophilus. The results provided conclusion on preferable usage of RAPD-PCR with the primer ERIC-1 for specific identification of S. thermophilus, and with the primer M13 for certification of natural isolates of this species at the strain level.     

<Emphasis Type="Italic">Paenibacillus bryophyllum</Emphasis> sp. nov., a nitrogen-fixing species isolated from <Emphasis Type="Italic">Bryophyllum pinnatum</Emphasis>

A nitrogen-fixing, endospore-forming bacterium, designated strain L201^T was isolated from the leaves of Bryophyllum pinnatum growing in South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain L201^T is affiliated with the genus Paenibacillus, and closely related to Paenibacillus albidus Q4-3^T (97.4%), Paenibacillus odorifer DSM 15391^T (97.3%) and Paenibacillus borealis DSM 13188^T (97.2%). The main fatty acids components was anteiso-C_15:0 (48.1%). The predominant isoprenoid quinone was MK-7. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The G+C content of strain L201^T was 43.9%. DNA–DNA relatedness between L201^T and the reference strain was 29.8%. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting and comparison of cellular fatty acids distinguished strain L201^T from the closely related Paenibacillus species. Based on these data, the novel species Paenibacillus bryophyllum sp. nov. is proposed, with the type strain L201^T(=?KCTC 33951 ^T?=?GDMCC 1.1251 ^T).     

<Emphasis Type="Italic">Streptomyces urticae</Emphasis> sp. nov., isolated from rhizosphere soil of <Emphasis Type="Italic">Urtica urens</Emphasis> L.

Two novel Gram-stain positive, spore-forming, aerobic actinomycetes, designated NEAU-PCY-1^T and NEAU-PCY-2, were isolated from rhizosphere soil of Urtica urens L. collected from Anshan, Liaoning Province, northeast China. The 16S rRNA gene sequence analysis showed that strains NEAU-PCY-1^T and NEAU-PCY-2 exhibited 99.8% similarity with each other and are closely related to Streptomyces abietis DSM 42080^T (98.2, 98.3%) and Streptomyces fildesensis DSM 41987^T (98.0, 98.1%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains formed a cluster with these two closely related species. Moreover, DNA–DNA hybridization results and some phenotypic, physiological and biochemical properties differentiated the two strains from their close relatives in the genus Streptomyces. Based on a polyphasic taxonomy study, strains NEAU-PCY-1^T and NEAU-PCY-2 are considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces urticae sp. nov. is proposed, with NEAU-PCY-1^T (=?DSM 105115^T?=?CCTCC AA 2017015^T) as the type strain.     

Evaluation of the Diversity of Probiotic <Emphasis Type="Italic">Bacillus</Emphasis>, <Emphasis Type="Italic">Clostridium</Emphasis>, and <Emphasis Type="Italic">Bifidobacterium</Emphasis> Using the Illumina-Based Sequencing Method

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.     

A RAPD fingerprinting of sibling species of the <Emphasis Type="Italic">Drosophila virilis</Emphasis> group

A comparative analysis of the sibling species of Drosophila virilis was performed by RAPD-PCR technique using a set of random primers. The degree of relatedness was studied by cluster analysis (UPGMA) and multidimensional scaling. The resulting pattern of species relationships contradicts the classical taxonomy. The main result of the cluster analysis is that D. virilis does not cluster with the remaining three species of its phylad, while according to multidimensional scaling, D. virilis is equidistant from all the species of its group, from both the species of its phylad and the species of the montana phylad. The montana phylad is extremely heterogeneous; moreover, the species D. littoralis, D. ezoana, and D. kanekoi appear to be closer to the virilis phylad than to the other species of the montana phylad, wherein these species are traditionally included. The phylogenetic relationships between the studied species discovered using RAPD fingerprinting comply with the results obtained using protein markers and quantitative traits.     

<Emphasis Type="Italic">Rhizobium hedysari</Emphasis> sp. nov., a novel species isolated from a root nodule of <Emphasis Type="Italic">Hedysarum multijugum</Emphasis> in China

A strain 5-1-2^T was isolated from a root nodule of Hedysarum multijugum collected from Zhangye city, Gansu province, north-west China. Phylogenetic analysis based on the 16S rRNA gene sequence and other housekeeping genes (recA and atpD) indicated that the strain represents a novel species in the genus Rhizobium close to the strain Rhizobium subbaraonis JC85^T with similarities of 98.27, 88.92 and 89.62%, respectively. Strain 5-1-2^T contained Q-10 as the predominant ubiquinone. Our results showed that the major fatty acids were feature 8 (C_18:1 ω7c and/or C_18:1 ω6c; 38.90%). In addition, the DNA–DNA hybridizations with the type strains R. subbaraonis JC85^T and Rhizobium halophytocola YC6881^T were 39.2 ± 2.1 and 44.3 ± 1.9, respectively. Therefore, a novel species Rhizobium hedysari sp. nov. is proposed, and 5-1-2^T (=CGMCC1.15677^T = NBRC112532^T) is designated as the type strain.     

<Emphasis Type="Italic">Salinispora</Emphasis><Emphasis Type="Italic">arenicola</Emphasis> from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes

The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected.     

Sequence Analysis and Comparative Bioinformatics Study of Camelysin Gene (<Emphasis Type="Italic">calY</Emphasis>) Isolated from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus strains have been widely used for the production of fibrinolytic enzymes having role in the treatment of cardiovascular disorders. Purification and overproduction of such enzymes has increased their usage in medical fields including metalloproteinases with the ability to degrade extracellular matrix (ECM). Camelysin, a neutral metalloproteinase has been isolated from different species of bacteria like Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis with fibrinolytic, collagenolytic and actin degradation activity. This project successfully demonstrated the presence of 734-bp coding DNA sequence (CDS) encoding a 20.72331 kDa camelysin gene in local strain of Bacillus thuringiensis containing a signal peptide with cleavage site between residues 19 and 20. The sequence was submitted to GenBank (KT023597) and the sequence showed high homology with the camelysin protein of closely related Bacillus species. The alignment of related proteins through ClustalW displayed difference of four amino acids (“Q” replaced by “P” at position 169 and at position 182–184, “NQE” replaced by “HLK”) in the isolated protein. Comparison including structural and functional analysis of camelysin sequences isolated from different Bacillus species was carried out using different bioinformatics tools and software. The information would help in better understanding the properties of camelysin protein and its role in pathogenicity and clinical treatments.     

<Emphasis Type="Italic">Rhodopirellula rosea</Emphasis> sp. nov., a novel bacterium isolated from an ark clam <Emphasis Type="Italic">Scapharca broughtonii</Emphasis>

A novel Gram-negative, motile, and ovoid-shaped strain, LHWP3^T, which belonged to the family Planctomycetaceae in the phylum Planctomycetes, was isolated from a dead ark clam Scapharca broughtonii collected during a mass mortality event on the south coast of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the isolate was most closely related to the type strain of Rhodopirellula baltica, with a shared 16S rRNA gene sequence similarity of 94.8%. The isolate grew optimally at 30°C in 4–6% (w/v) NaCl, and at pH 7. The major isoprenoid quinone was menaquinone-6 (MK-6). The dominant polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and unidentified polar lipids. The predominant cellular fatty acids were C_16:0, C_18:1 ω9c, and C_18:0. The genomic DNA G+C content of strain LHWP3^T was 53.0 mol%. Based on polyphasic taxonomic analyses, strain LHWP3^T should be classified as a novel species in the genus Rhodopirellula in the family Planctomycetaceae, for which the name Rhodopirellula rosea sp. nov. is proposed. The type strain is LHWP3^T (=KACC 15560^T =JCM 17759^T).     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.     

<Emphasis Type="Italic">Methylophilaceae</Emphasis> and <Emphasis Type="Italic">Hyphomicrobium</Emphasis> as target taxonomic groups in monitoring the function of methanol-fed denitrification biofilters in municipal wastewater treatment plants

Molecular monitoring of bacterial communities can explain and predict the stability of bioprocesses in varying physicochemical conditions. To study methanol-fed denitrification biofilters of municipal wastewater treatment plants, bacterial communities of two full-scale biofilters were compared through fingerprinting and sequencing of the 16S rRNA genes. Additionally, 16S rRNA gene fingerprinting was used for 10-week temporal monitoring of the bacterial community in one of the biofilters. Combining the data with previous study results, the family Methylophilaceae and genus Hyphomicrobium were determined as suitable target groups for monitoring. An increase in the relative abundance of Hyphomicrobium-related biomarkers occurred simultaneously with increases in water flow, NO _x ^? load, and methanol addition, as well as a higher denitrification rate, although the dominating biomarkers linked to Methylophilaceae showed an opposite pattern. The results indicate that during increased loading, stability of the bioprocess is maintained by selection of more efficient denitrifier populations, and this progress can be analyzed using simple molecular fingerprinting.     

Characterization of <Emphasis Type="Italic">lpaH2</Emphasis> gene corresponding to lipopeptide synthesis in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> HAB-2

Background

Bacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4′-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide.

Results

A sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2.

Conclusion

Few studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.