Journal of Microbiology

Volume 47(4); August 2009

Research Support, Non-U.S. Gov'ts

Comparison of the Bacterial Community and Characterization of Plant Growth-Promoting Rhizobacteria from Different Genotypes of Chrysopogon zizanioides (L.) Roberty (Vetiver) Rhizospheres: Juliana Mendes Monteiro , Renata Estebanez Vollu , Marcia Reed Rodrigues Coelho , Celuta Sales Alviano , Arie Fitzgerald Blank , Lucy Seldin; J. Microbiol. 2009;47(4):363-370. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0048-3

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Abstract: Molecular approaches [PCR-denaturing gradient gel electrophoresis (DGGE)] were used to determine whether three different vetiver (Chrysopogon zizanioides) genotypes, commercially used in Brazil and considered economically important over the world, select specific bacterial populations to coexist in their rhizospheres. DGGE profiles revealed that the predominant rhizospheric bacterial community hardly varies regarding the vetiver genotype. Moreover, using traditional cultivation methods, bacterial strains were isolated from the different rhizospheres. Colonies presenting different morphologies (83) were selected for determining their potential for plant growth promotion. More than half of the strains tested (57.8%) were amplified by PCR using nifH-based primers, specific for the enzyme nitrogenase reductase. The production of siderophores was observed in 88% of the strains, while the production of antimicrobial substances was detected in only 14.5% of the isolates when Micrococcus sp. was used as the indicator strain. Production of indole-3-acetic acid and the solubilization of phosphate were observed in 55.4% and 59% of the isolates, respectively. In total, 44 strains (53%) presented at least three characteristics of plant growth promotion and were submitted to amplified ribosomal DNA restriction analysis. Twenty-four genetic groups were formed at 100% similarity and one representative of each group was selected for their identification by partial 16S rRNA gene sequencing. They were affiliated with the genera Acinetobacter, Comamonas, Chryseobacterium, Klebsiella, Enterobacter, Pantoea, Dyella, Burkholderia, or Pseudomonas. These strains can be considered of great importance as possible biofertilizers in vetiver.

Molecular Analysis of Prokaryotic Diversity in the Deep Subsurface of the Former Homestake Gold Mine, South Dakota, USA: Gurdeep Rastogi , Larry D. Stetler , Brent M. Peyton , Rajesh K. Sani; J. Microbiol. 2009;47(4):371-384. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-008-0249-1

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Abstract: A culture-independent molecular phylogenetic analysis was carried out to study the prokaryotic diversity in two soil samples collected from the subsurface (1.34 km depth) of the former Homestake gold mine, Lead, South Dakota, USA at two sites, the Ross shaft and number 6 Winze. Microbial community analyses were performed by cloning and sequencing of 16S rRNA genes retrieved directly from soil samples. Geochemical characterization of soils revealed high amount of toxic metals such as As, Cd, Co, Cr, Cu, Ni, Pb, Zn, and U at both the sites. Phylogenetic analyses showed that soil samples were predominantly composed of phylotypes related to phylum Proteobacteria. Other phyla detected in libraries were Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Chlorobi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Verrucomicrobia, and candidate divisions OP10 and TM7. The majority (>95%) of the phylotypes retrieved in the libraries were most closely related to environmental sequences from yet-uncultured bacteria representing a hitherto unidentified diversity. The archaeal communities at both the sites exhibited lower diversity and were most closely affiliated to uncultivated species within the Crenarchaeota. Results showed the existence of diverse microbial populations in deep subsurface environment of the Homestake gold mine. Statistical analyses demonstrated that each site harbored phylogenetically distinct microbial populations that were more diverse at Ross site compare to winze site.

Evaluation of Antagonistic Activities of Bacillus subtilis and Bacillus licheniformis Against Wood-Staining Fungi: In Vitro and In Vivo Experiments: Natarajan Velmurugan , Mi Sook Choi , Sang-Sub Han , Yang-Soo Lee; J. Microbiol. 2009;47(4):385-392. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0018-9

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Abstract: The antifungal activity of bacterial strains Bacillus subtilis EF 617317 and B. licheniformis EF 617325 was demonstrated against sapstaining fungal cultures Ophiostoma flexuosum, O. tetropii, O. polonicum, and O. ips in both in vitro and in vivo conditions. The crude active supernatant fractions of 7 days old B. subtilis and B. licheniformis cultures inhibited the growth of sapstaining fungi in laboratory experiments. Thermostability and pH stability of crude supernatants were determined by series of experiments. FT-IR analysis was performed to confirm the surface structural groups of lipoproteins present in the crude active supernatant. Partial purification of lipopeptides present in the crude supernatant was done by using Cellulose anion exchange chromatography and followed by Sephadex gel filtration chromatography. Partially purified compounds significantly inhibited the sapstaining fungal growth by in vitro analysis. The lipopeptides responsible for antifungal activity were identified by electrospray ionization mass spectrometry after partial purification by ion exchange and gel filtration chromatography. Four major ion peaks were identified as m/z 1023, 1038, 1060, and 1081 in B. licheniformis and 3 major ion peaks were identified as m/z 1036, 1058, and 1090 in B. subtilis. In conclusion, the partially purified lipopeptides may belong to surfactin and iturin family. In vivo analysis for antifungal activity of lipopeptides on wood was conducted in laboratory. In addition, the potential of extracts for fungal inhibition on surface and internal part of wood samples were analyzed by scanning electron microscopy.

Endophytic Bacterial Diversity in Grapevine (Vitis vinifera L.) Leaves Described by 16S rRNA Gene Sequence Analysis and Length Heterogeneity-PCR: Daniela Bulgari , Paola Casati , Lorenzo Brusetti , Fabio Quaglino , Milena Brasca , Daniele Daffonchio , Piero Attilio Bianco; J. Microbiol. 2009;47(4):393-401. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0082-1

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Abstract

Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma- infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

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Identification and Characterization of Metagenomic Fragments from Tidal Flat Sediment: Byung Kwon Kim , Yoon-Dong Park , Hyun-Myung Oh , Jongsik Chun; J. Microbiol. 2009;47(4):402-410. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0099-5

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Abstract: Phylogenetic surveys based on cultivation-independent methods have revealed that tidal flat sediments are environments with extensive microbial diversity. Since most of prokaryotes in nature cannot be easily cultivated under general laboratory conditions, our knowledge on prokaryotic dwellers in tidal flat sediment is mainly based on the analysis of metagenomes. Microbial community analysis based on the 16S rRNA gene and other phylogenetic markers has been widely used to provide important information on the role of microorganisms, but it is basically an indirect means, compared with direct sequencing of metagenomic DNAs. In this study, we applied a sequence-based metagenomic approach to characterize uncultivated prokaryotes from tidal flat sediment. Two large-insert genomic libraries based on fosmid were constructed from tidal flat metagenomic DNA. A survey based on end-sequencing of selected fosmid clones resulted in the identification of clones containing 274 bacterial and 16 archaeal homologs in which majority were of proteobacterial origins. Two fosmid clones containing large metagenomic DNAs were completely sequenced using the shot- gun method. Both DNA inserts contained more than 20 genes encoding putative proteins which implied their ecological roles in tidal flat sediment. Phylogenetic analyses of evolutionary conserved proteins indicate that these clones are not closely related to known prokaryotes whose genome sequence is known, and genes in tidal flat may be subjected to extensive lateral gene transfer, notably between domains Bacteria and Archaea. This is the first report demonstrating that direct sequencing of metagenomic gene library is useful in underpinning the genetic makeup and functional roles of prokaryotes in tidal flat sediments.

Impact of Some Environmental Factors on Growth and Production of Ochratoxin A of/by Aspergillus tubingensis, A. niger, and A. carbonarius Isolated from Moroccan Grapes: Atar Selouane , Driss Bouya , Ahmed Lebrihi , C. Decock , Amina Bouseta; J. Microbiol. 2009;47(4):411-419. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-008-0236-6

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Abstract: The effects of temperature, water activity (aw), incubation time, and their combinations on radial growth and ochratoxin A (OTA) production of/by eight Aspergillus niger aggregate strains (six A. tubingensis and two A. niger) and four A. carbonarius isolated from Moroccan grapes were studied. Optimal conditions for the growth of most studied strains were shown to be at 25°C and 0.95 aw. No growth was observed at 10°C regardless of the water activity and isolates. The optimal temperature for OTA production was in the range of 25°C~30°C for A. carbonarius and 30°C~37°C for A. niger aggregate. The optimal aw for toxin production was 0.95~0.99 for A. carbonarius and 0.90~0.95 for A. niger aggregate. Mean OTA concentration produced by all the isolates of A. niger aggregate tested at all sampling times shows that maximum amount of OTA (0.24 µg/g) was produced at 37°C and 0.90 aw. However, for A. carbonarius, mean maximum amounts of OTA (0.22 µg/g) were observed at 25°C and 0.99 aw. Analysis of variance showed that the effects of all single factors (aw, isolate, temperature and incubation time) and their interactions on growth and OTA production were highly significant.

Effect of Fungal Pellet Morphology on Enzyme Activities Involved in Phthalate Degradation: Young-Mi Kim , Hong-Gyu Song; J. Microbiol. 2009;47(4):420-424. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0051-8

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Abstract: Pellet size of white rot fungus, Pleurotus ostreatus may affect the secretion of its degradative enzymes and accompanying biodegrading capability, but could be controlled by several physical culture conditions in liquid culture. The pellet size of P. ostreatus was affected by the volume of inoculum, flask, and medium, but the agitation speed was the most important control factor. At the lower agitation speed of 100 rpm, the large pellets were formed and the laccase activity was higher than that of small pelleted culture at 150 rpm, which might be due to loose intrapellet structure. However, the biodegradation rates of benzylbutylphthalate and dimethylphthalate were higher in the small pelleted culture, which indicated the involvement of other degradative enzyme rather than laccase. The activity of esterase which catalyzes the nonphenolic compounds before the reaction of ligninolytic enzymes was higher in the small pelleted culture, and coincided with the degradation pattern of phthalates. This study suggests the optimization of pellet morphology and subsequent secretion of degradative enzymes is necessary for the efficient removal of recalcitrants by white rot fungi.

Chrysosporium pseudomerdarium Produces Gibberellins and Promotes Plant Growth: Muhammad Hamayun , Sumera Afzal Khan , Ilyas Iqbal , Chae-In Na , Abdul Latif Khan , Young-Hyun Hwang , In-Jung Lee; J. Microbiol. 2009;47(4):425-430. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0268-6

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Abstract: We isolated 10 endophytic fungi from the roots of drought stressed soybean cultivar Hwangkeumkong and bioassyed on waito-c rice and soybean seedlings, in order to identify plant growth-promoting fungi. The fungal isolate D-2-1 provided the best result for plant height and biomass promotion as compared to wild type Gibberella fujikuroi. The D-2-1 culture filtrate (CF) was analyzed for the presence of gibberellins (GAs) and it was observed that all physiologically active GAs, especially gibberellic acid, were present in higher amounts (GA1, 0.24 ng/ml; GA3, 8.99 ng/ml; GA4, 2.58 ng/ml and GA7, 1.39 ng/ml) in conjunction with physiologically inactive GA5, GA9, GA15, GA19, and GA24. The fungal isolate D-2-1 was identified as a new strain of Chrysosporium pseudomerdarium through phylogenetic analysis of 18S rDNA sequence. Plant growth promotion and GAs production capacity of genus Chrysosporium have been reported for the first time in this study.

Identification and Characterization of a Class III Chitin Synthase Gene of Moniliophthora perniciosa, the Fungus That Causes Witches' Broom Disease of Cacao: Catiane S. Souza , Bruno M. Oliveira , Gustavo G. L. Costa , Albert Schriefer , Alessandra Selbach-Schnadelbach , Ana Paula T. Uetanabaro , Carlos P. Pirovani , Goncalo A. G. Pereira , Alex G. Taranto , Julio Cezar de M. Cascardo , Aristoteles Goes-Neto; J. Microbiol. 2009;47(4):431-440. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-008-0166-3

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Abstract: Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches’ broom disease of cacao.

Molecular Diversity of Chrysoviruses in Korean Isolates of A New Fungal Species, Cryphonectria nitschkei: Jung-Mi Kim , Jung-Ae Kim , Jin-Ah Park , Seung-Moon Park , Byeong-Jin Cha , Moon-Sik Yang , Dae-Hyuk Kim; J. Microbiol. 2009;47(4):441-447. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0206-7

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Abstract: Genetic diversity of the chrysovirus within the four fungal strains was analyzed by comparing the full- length sequences of cloned chrysoviral genes encoding the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP). Because the morphological characteristics of four chrysovirus-infected Cryphonectria spp. strains were different, strain identification was conducted via sequence comparison of the internal transcribed spacers (ITSs) of the fungal rRNA gene. Phylogenic analysis of the ITS regions revealed that the four strains were closely clustered with the reference strain of Cryphonectria nitschkei, while they were more distantly related to other common Cryphonectria species, indicating that they were likely C. nitschkei. Sequence comparison among chrysoviruses from Korean C. nitschkei strains revealed that similarities of the RdRp and CP genes ranged from 98% to 100% and from 95% to 100%, respectively, at the protein level. Their corresponding nucleotide sequences showed 97% to 100% and 84% to 100% identities, respectively. Compared to RdRp, the CP gene had more divergence, suggesting the presence of genes possessing different evolutionary rates within the chrysovirus genome. Sequence comparisons with other known chrysoviruses showed that the four Korean chrysoviruses were clustered together at the next lineage level. Discovering why two strains (bs131 and bs132) containing identical ITS sequences and chrysoviruses display different phenotypes should prove interesting.

Transmission of Methylobacterium mesophilicum by Bucephalogonia xanthophis for Paratransgenic Control Strategy of Citrus Variegated Chlorosis: Cláudia Santos Gai , Paulo Teixeira Lacava , Maria Carolina Quecine , Marie-Christine Auriac , João Roberto Spotti Lopes , Welington Luiz Araújo , Thomas Albert Miller , João Lúcio Azevedo; J. Microbiol. 2009;47(4):448-454. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-008-0303-z

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Abstract: Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.

Journal Article

Gene Expression Profile of Helicobacter pylori in Response to Growth Temperature Variation: Yue-hua Han , Wen-zhong Liu , Yao-zhou Shi , Li-qiong Lu , Shu-dong Xiao , Qing-hua Zhang; J. Microbiol. 2009;47(4):455-465. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0003-3

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Abstract: A Helicobacter pylori whole-genome DNA microarray was constructed to study expression profiles of H. pylori in response to a sudden temperature transfer from 37°C to 20°C. The expression level of the genome at each of four time points (15, 30, 60, and 120 min) after temperature downshift was compared with that just before cold treatment. Globally, 10.2% (n=167) of the total predicted H. pylori genes (n=1636) represented on the microarray were significantly differentially expressed (p<0.05) over a 120 min period after shift to low temperature. The expression profiles of the differentially expressed genes were grouped, and their expression patterns were validated by quantitative real-time PCR. Up-regulated genes mainly included genes involved in energy metabolism and substance metabolism, cellular processes, protein fate, ribosomal protein genes, and hypothetical protein genes, which indicate the compensational responses of H. pylori to temperature downshift. Those genes play important roles in adaption to temperature downshift of H. pylori. Down-regulation of DNA metabolism genes and cell envelope genes and cellular processes genes may reflect damaged functions under low temperature, which is unfavorable to bacterial infection and propagation. Overall, this time-course study provides new insights into the primary response of H. pylori to a sudden temperature downshift, which allow the bacteria to survive and adapt to the new host environment.

Research Support, Non-U.S. Gov'ts

Cloning and Molecular Characterization of a Novel Rolling-Circle Replicating Plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1: Ming Shun Li , Jong Yul Roh , Xueying Tao , Zi Niu Yu , Zi Duo Liu , Qin Liu , Hong Guang Xu , Hee Jin Shim , Yang-Su Kim , Yong Wang , Jae Young Choi , Yeon Ho Je; J. Microbiol. 2009;47(4):466-472. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0020-2

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Abstract: Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki K1 which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pK1S-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pK1S-1, ORF2 (MobK1) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8~25.2%) with the Rep protein coded by RCR plasmids, however. The putative double- strand origin of replication (dso) and single-strand origin of replication (sso) of pK1S-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pK1S-1, seven subclones were contructed in the B. thuringiensis ori-negative pHT1K vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (Rep1K), dso and ORF4, exhibited replication ability. These findings identified pK1S-1 as a new RCR group VII plasmid, and determined its replication region.

Identification and Characterization of Acetyl-CoA Carboxylase Gene Cluster in Streptomyces toxytricini: Atanas V. Demirev , Ji Seon Lee , Bhishma R. Sedai , Ivan G. Ivanov , Doo Hyun Nam; J. Microbiol. 2009;47(4):473-478. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0135-5

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Abstract: The gene locus for acetyl-CoA carboxylase (ACC) involved in the primary metabolism was identified from the genomic library of Streptomyces toxytricini which produces a lipase inhibitor lipstatin. The 7.4 kb cloned gene was comprised of 5 ORFs including accD1, accA1, hmgL, fadST1, and stsF. In order to confirm the biochemical characteristics of AccA1, the gene was overexpressed in Escherichia coli cells, and the recombinant protein was purified through Ni2+ affinity chromatography. Because most of the expressed AccA1 was biotinylated by host E. coli BirA in the presence of D-biotin, the non-biotinylated apo-AccA1 was purified after gene induction without D-biotin, followed by exclusion of holo-AccA1 using streptavidin beads. The separated apo-AccA1 was post-translationally biotinylated by S. toxytricini biotin apo-protein ligase (BPL) in a time- and enzyme-dependent manner. This result supports that this gene cluster of S. toxytricini encodes the functional ACC enzyme subunits to be biotinylated.

Effect of Iron on Cytolysin A Expression in Salmonella enterica serovar Typhi: Jinghua Cui , Honghua Piao , Shen Jin , Hee Sam Na , Yeongjin Hong , Hyon E Choy , Phil Youl Ryu; J. Microbiol. 2009;47(4):479-485. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0039-4

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Abstract: Previously, a novel protein ClyA (Cytolysin A) has been identified in Escherichia coli K-12, Salmonella enterica serovars Typhi and Paratyphi A and Shigella. Salmonella spp. synthesize substantial amounts of ClyA upon infection of the human host, although the mechanism by which ClyA is induced in vivo is unclear. Since environmental signals could control the expression of virulence determinants, ClyA expression in S. Typhi Ty2 was tested by Western blotting in the presence of normal pooled human serum (NPS). The level of ClyA expression increased in the presence of NPS in a concentration-dependent manner. RPMI 1640 medium similarly induced ClyA expression. ClyA expression was inversely proportional to the concentration of iron in RPMI medium. Therefore, we speculated that iron inhibited the expression of ClyA in S. Typhi Ty2, and free iron depletion may be one of the causes of S. Typhi-mediated induction of ClyA in vivo. Transcription from a clyA-lacZ fusion gene decreased as iron concentration increased, but not as significantly as the ClyA protein expression. It is concluded that the regulatory effect of iron on clyA expression is mainly at translational level.

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