Journal of Microbiology

Journal Article

Cultivation of Diverse Novel Marine Bacteria from Deep Ocean Sediment Using Spent Culture Supernatant of Ca. Bathyarchaeia Enrichment.: Sidra Erum Ishaq, Tariq Ahmad, Lewen Liang, Ruize Xie, Tiantian Yu, Yinzhao Wang, Fengping Wang; J. Microbiol. 2024;62(8):611-625. Published online July 10, 2024; DOI: https://doi.org/10.1007/s12275-024-00145-w

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Abstract: Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.

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

Statistical experimental design optimization of rhamsan gum production by Sphingomonas sp. CGMCC 6833: Xiao-Ying Xu , Shu-Hao Dong , Sha Li , Xiao-Ye Chen , Ding Wu , Hong Xu; J. Microbiol. 2015;53(4):272-278. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-3662-2

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11 Citations

Abstract: Rhamsan gum is a type of water-soluble exopolysaccharide produced by species of Sphingomonas bacteria. The optimal fermentation medium for rhamsan gum production by Sphingomonas sp. CGMCC 6833 was explored definition. Single-factor experiments indicate that glucose, soybean meal, K2HPO4 and MnSO4 compose the optimal medium along with and initial pH 7.5. To discover ideal cultural conditions for rhamsan gum production in a shake flask culture, response surface methodology was employed, from which the following optimal ratio was derived: 5.38 g/L soybean meal, 5.71 g/L K2HPO4 and 0.32 g/L MnSO4. Under ideal fermentation rhamsan gum yield reached 19.58 g/L ?1.23 g/L, 42.09% higher than that of the initial medium (13.78 g/L ? 1.38 g/L). Optimizing the fermentation medium results in enhanced rhamsan gum production.

Morphological Structure of Propagules and Electrophoretic Karyotype Analysis of False Smut Villosiclava virens in Rice: Rongtao Fu , Lei Ding , Jun Zhu , Ping Li , Ai-ping Zheng; J. Microbiol. 2012;50(2):263-269. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1456-3

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23 Citations

Abstract: The target pathogen Villosiclava virens (teleomorph: claviceps oryzae-sativae) was isolated from the infected rice, where it caused false smut. In our study, the forming processes of the chlamydospores, chlamydospore balls, conidiospores, and secondary conidiospores during the asexual reproduction were observed more precisely and in greater detail than previous descriptions. The microstructure of the infected rice kernel showed that the outer dense chlamydospores piled around the false smut balls grown on XBZ medium; moreover the sclerotia consisting of dense mycelium were found. The different morphology was observed across the different growing conditions. In addition, we observed the nuclear numbers of both the conidiospores and hyphae using 4′,6-diamidino-2-phenylindole (DAPI) staining. Because the fungus has small chromosomes and the numbers were not previously known, we analyzed the electrophoretic karyotype using a pulsed field gel electrophoresis (PFGE) technique. The results showed that V. virens has at least 10 chromosomes ranging in size from 0.6 kb to 6 Mb. The V. virens genome size is estimated to be 23 Mb. Here, we report the morphological characteristics of the fungus and the process of asexual spores forming asexual propagules, along with the first analyze the molecular karyotype of V. virens. These
results
supply a foundation for further study of the pathogenicity and biology of this devastating pathogen.

Review

Biosynthesis, Modification, and Biodegradation of Bacterial Medium-Chain-Length Polyhydroxyalkanoates: Do Young Kim , Hyung Woo Kim , Moon Gyu Chung , Young Ha Rhee; J. Microbiol. 2007;45(2):87-97.; DOI: https://doi.org/2528 [pii]

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Abstract: Medium-chain-length polyhydroxyalkanoates (MCL-PHAs), which have constituents with a typical chain length of C6-C14, are polyesters that are synthesized and accumulated in a wide variety of Gram-negative bacteria, mainly pseudomonads. These biopolyesters are promising materials for various applications because they have useful mechanical properties and are biodegradable and biocompatible. The versatile metabolic capacity of some Pseudomonas spp. enables them to synthesize MCL-PHAs that contain various functional substituents; these MCL-PHAs are of great interest because these functional groups can improve the physical properties of the polymers, allowing the creation of tailor-made products. Moreover, some functional substituents can be modified by chemical reactions to obtain more useful groups that can extend the potential applications of MCL-PHAs as environmentally friendly polymers and functional biomaterials for use in biomedical fields. Although MCL-PHAs are water-insoluble, hydrophobic polymers, they can be degraded by microorganisms that produce extracellular MCL-PHA depolymerase. MCL-PHA-degraders are relatively uncommon in natural environments and, to date, only a limited number of MCL-PHA depolymerases have been investigated at the molecular level. All known MCL-PHA depolymerases share a highly significant similarity in amino acid sequences, as well as several enzymatic characteristics. This paper reviews recent advances in our knowledge of MCL-PHAs, with particular emphasis on the findings by our research group.

Research Support, Non-U.S. Gov't

Molecular Characterization of Extracellular Medium-chain-length Poly(3-hydroxyalkanoate) Depolymerase Genes from Pseudomonas alcaligenes Strains: Do Young Kim , Hyun Chul Kim , Sun Young Kim , Young Ha Rhee; J. Microbiol. 2005;43(3):285-294.; DOI: https://doi.org/2211 [pii]

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Abstract: A bacterial strain M4-7 capable of degrading various polyesters, such as poly(e-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ_PalM4-7) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ_PalM4-7 was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35^oC. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ_PalLB19) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M_r of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ_PalM4-7) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced M_r of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ_PalLB19 and the PhaZ_PalM4-7 commonly share the lipase box, GISSG, in their catalytic domains, and utilize ^111Asn and ^110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.

Evaluation of the EF-18 Agar-Hydrophobic Grid Membrane Filter (HGMF) Method to Isolate Salmonella from Poultry Products: Rosa Capita , Maite Alvarez-Astorga , Carlos Alonso-Calleja , Maria del Camino , Garcia-Fernandez , Benito Moreno; J. Microbiol. 2001;39(3):202-205.

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Abstract: The EF-18 agar/hydrophobic grid membrane filter (EF18/HGMF) method was evaluated for the isolation of Salmonella in naturally contaminated chicken carcasses, chicken parts (legs, wings and giblets) and processed chicken products (sausages and hamburgers). Percentages of false positive results for Salmonella (colonies with a similar morphology to those of Salmonella) were 78.75, 81.67 and 80% for carcasses, chicken parts and processed chicken products, respectively. The bacterial isolates that caused false positive reactions using this method were identified as Proteus mirabilis (70.85%), Citrobacter freundii (15.25%), Klebsiella ozaenae (5.83%), Hafnia alvei (4.48%), Escherichia coli (2.69%) and Enterobacter aerogenes (0.90%). The data obtained in this study suggest that the EF-18/HGMF method is not sufficiently selective or specific for isolating Salmonella from meat and chicken products.

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