Journal of Microbiology

Journal Article

Antimicrobial Efficacy of Allium cepa and Zingiber officinale Against the Milk‑Borne Pathogen Listeria monocytogenes: Abirami Arasu , Nagaram Prabha , Durga Devi , Praveen Kumar Issac , Khaloud Mohammed Alarjani , Dunia A. Al Farraj , Reem A. Aljeidi , Dina S. Hussein , Magesh Mohan , Jehad Zuhair Tayyeb , Ajay Guru , Jesu Arockiaraj; J. Microbiol. 2023;61(11):993-1011. Published online December 4, 2023; DOI: https://doi.org/10.1007/s12275-023-00086-w

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Listeria monocytogenes is an important food-borne pathogen that causes listeriosis and has a high case fatality rate despite its low incidence. Medicinal plants and their secondary metabolites have been identified as potential antibacterial substances, serving as replacements for synthetic chemical compounds. The present studies emphasize two significant medicinal plants, Allium cepa and Zingiber officinale, and their efficacy against L. monocytogenes. Firstly, a bacterial isolate was obtained from milk and identified through morphology and biochemical reactions. The species of the isolate were further confirmed through 16S rRNA analysis. Furthermore, polar solvents such as methanol and ethanol were used for the extraction of secondary metabolites from A. cepa and Z. officinale. Crude phytochemical components were identified using phytochemical tests, FTIR, and GC–MS. Moreover, the antibacterial activity of the crude extract and its various concentrations were tested against L. monocytogenes. Among all, A. cepa in methanolic extracts showed significant inhibitory activity. Since, the A. cepa for methanolic crude extract was used to perform autography to assess its bactericidal activity. Subsequently, molecular docking was performed to determine the specific compound inhibition. The docking results revealed that four compounds displayed strong binding affinity with the virulence factor Listeriolysin-O of L. monocytogenes. Based on the above results, it can be concluded that the medicinal plant A. cepa has potential antibacterial effects against L. monocytogenes, particularly targeting its virulence.

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Cultural Perspectives on the Sustainable Use and Added Value of Plant-Based Food Dyes—A Case Study from Bulgaria
Mihail Chervenkov, Teodora Ivanova, Yulia Bosseva, Dessislava Dimitrova
Sustainability.2024; 16(20): 9049. CrossRef

Review

Temperature Matters: Bacterial Response to Temperature Change: Seongjoon Moon , Soojeong Ham , Juwon Jeong , Heechan Ku , Hyunhee Kim , Changhan Lee; J. Microbiol. 2023;61(3):343-357. Published online April 3, 2023; DOI: https://doi.org/10.1007/s12275-023-00031-x

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Abstract

Temperature is one of the most important factors in all living organisms for survival. Being a unicellular organism, bacterium requires sensitive sensing and defense mechanisms to tolerate changes in temperature. During a temperature shift, the structure and composition of various cellular molecules including nucleic acids, proteins, and membranes are affected. In addition, numerous genes are induced during heat or cold shocks to overcome the cellular stresses, which are known as heat- and cold-shock proteins. In this review, we describe the cellular phenomena that occur with temperature change and bacterial responses from a molecular perspective, mainly in Escherichia coli.

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Journal Articles

Superantigen SpeA attenuates the biofilm forming capacity of Streptococcus pyogenes: Anshu Babbar , Israel Barrantes , Dietmar H. Pieper , Andreas Itzek; J. Microbiol. 2019;57(7):626-636. Published online June 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8648-z

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Abstract

Beta haemolytic Group A streptococcus (GAS) or Streptococcus pyogenes are strict human pathogens responsible for mild to severe fatal invasive infections. Even with enormous number of reports exploring the role of S. pyogenes exotoxins in its pathogenesis, inadequate knowledge on the biofilm process and the potential role of exotoxins in bacterial dissemination from matured biofilms has been a hindrance in development of effective and targeted treatments. Therefore, the present study was aimed in investigating the uncharted role of these exotoxins in biofilm process. Through our study the putative role of ciaRH in the SpeA dependent ablation of biofilm formation could be speculated and thus helping in bacterial dissemination. The seed-dispersal effect of SpeA was time and concentration dependent and seen to be consistent within various streptococcal species. Transcriptome analysis of SpeA treated S. pyogenes biofilms revealed the involvement of many transcriptional regulators (ciaRH) and response genes (luxS, shr, shp, SPy_0572), hinting towards specific mechanisms underlying the dispersal effect by SpeA. This finding opens up a discussion towards understanding a new mechanism involved in the pathogenesis of Streptococcus pyogenes and might help in understanding the bacterial infections in a better way.

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Pathomolecular epidemiology, antimicrobial resistance, and virulence genes of Streptococcus dysgalactiae subsp. equisimilis isolates from slaughtered pigs in India
Sagar M Patel, Monalisa Sahoo, Jigarji Chaturji Thakor, Dinesh Murali, Pradeep Kumar, Rajendra Singh, Karam Pal Singh, G Saikumar, Chandrakanta Jana, Shailesh Kumar Patel, Akash B Mote, Ravichandran Karthikeyan, Rajesh Kumar Vandre, Jitendra Kumar Biswal,
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T. M. Rumynska, A. R. Hural, Y. T. Konechnyi, R. B. Vynnytska, A. V. Lozynskyi, Y. T. Salyha, O. P. Korniychuk, R.B. Lesyk
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Gilbert Donders, Peter Greenhouse, Francesca Donders, Ulrike Engel, Jorma Paavonen, Werner Mendling
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Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light: Itzel Margarita Córdova-Alcántara , Diana Laura Venegas-Cortés , María Ángeles Martínez-Rivera , Néstor Octavio Pérez , Aida Verónica Rodriguez-Tovar; J. Microbiol. 2019;57(6):485-497. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8637-2

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Abstract

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.

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Negative interaction of Staphylococcus aureus on Fusarium falciforme growth ocular isolates in an in vitro mixed biofilm
Luis Antonio Bautista-Hernández, José Luis Gómez-Olivares, Beatriz Buentello-Volante, Alfredo Dominguez-Lopez, Yonathan Garfias, María Cristina Acosta-García, Rosa Paulina Calvillo-Medina, Victor Manuel Bautista-de Lucio
Microbial Pathogenesis.2019; 135: 103644. CrossRef

Reviews

REVIEW] Recent paradigm shift in the assembly of bacterial tripartite efflux pumps and the type I secretion system: Inseong Jo , Jin-Sik Kim , Yongbin Xu , Jaekyung Hyun , Kangseok Lee , Nam-Chul Ha; J. Microbiol. 2019;57(3):185-194. Published online February 26, 2019; DOI: https://doi.org/10.1007/s12275-019-8520-1

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Abstract

Tripartite efflux pumps and the type I secretion system of Gram-negative bacteria are large protein complexes that span the entire cell envelope. These complexes expel antibiotics and other toxic substances or transport protein toxins from bacterial cells. Elucidating the binary and ternary complex structures at an atomic resolution are crucial to understanding the assembly and working mechanism. Recent advances in cryoelectron microscopy along with the construction of chimeric proteins drastically shifted the assembly models. In this review, we describe the current assembly models from a historical perspective and emphasize the common assembly mechanism for the assembly of diverse tripartite pumps and type I secretion systems.

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Molecular mechanisms of antibiotic resistance revisited
Elizabeth M. Darby, Eleftheria Trampari, Pauline Siasat, Maria Solsona Gaya, Ilyas Alav, Mark A. Webber, Jessica M. A. Blair
Nature Reviews Microbiology.2023; 21(5): 280. CrossRef
Permeation of Fosfomycin through the Phosphate-Specific Channels OprP and OprO of Pseudomonas aeruginosa
Vinaya Kumar Golla, Claudio Piselli, Ulrich Kleinekathöfer, Roland Benz
The Journal of Physical Chemistry B.2022; 126(7): 1388. CrossRef
Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability
Eric Mandela, Christopher J Stubenrauch, David Ryoo, Hyea Hwang, Eli J Cohen, Von L Torres, Pankaj Deo, Chaille T Webb, Cheng Huang, Ralf B Schittenhelm, Morgan Beeby, JC Gumbart, Trevor Lithgow, Iain D Hay
eLife.2022;[Epub] CrossRef
Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria
Ilyas Alav, Jessica Kobylka, Miriam S. Kuth, Klaas M. Pos, Martin Picard, Jessica M. A. Blair, Vassiliy N. Bavro
Chemical Reviews.2021; 121(9): 5479. CrossRef
Biotechnological applications of type 1 secretion systems
Zohreh Pourhassan N., Sander H.J. Smits, Jung Hoon Ahn, Lutz Schmitt
Biotechnology Advances.2021; 53: 107864. CrossRef
Protein-Protein Interactions in the Cytoplasmic Membrane of Escherichia coli: Influence of the Overexpression of Diverse Transporter-Encoding Genes on the Activities of PTS Sugar Uptake Systems
Mohammad Aboulwafa, Zhongge Zhang, Milton H. Saier Jr.
Microbial Physiology.2020; 30(1-6): 36. CrossRef
Perspectives towards antibiotic resistance: from molecules to population
Joon-Hee Lee
Journal of Microbiology.2019; 57(3): 181. CrossRef

MINIREVIEW] High-resolution imaging of the microbial cell surface: Ki Woo Kim; J. Microbiol. 2016;54(11):703-708. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6348-5

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Abstract

Microorganisms, or microbes, can function as threatening pathogens that cause disease in humans, animals, and plants; however, they also act as litter decomposers in natural ecosystems. As the outermost barrier and interface with the environment, the microbial cell surface is crucial for cell-to-cell communication and is a potential target of chemotherapeutic agents. Surface ultrastructures of microbial cells have typically been observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Owing to its characteristics of low-temperature specimen preparation and superb resolution (down to 1 nm), cryo-field emission SEM has revealed paired rodlets, referred to as hydrophobins, on the cell walls of bacteria and fungi. Recent technological advances in AFM have enabled high-speed live cell imaging in liquid at the nanoscale level, leading to clear visualization of celldrug interactions. Platinum-carbon replicas from freeze-fractured fungal spores have been observed using transmission electron microscopy, revealing hydrophobins with varying dimensions. In addition, AFM has been used to resolve bacteriophages in their free state and during infection of bacterial cells. Various microscopy techniques with enhanced spatial resolution, imaging speed, and versatile specimen preparation are being used to document cellular structures and events, thus addressing unanswered biological questions.

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Yu Sun, Can Cao, Yilin Peng, Xuyao Dai, Xiaoke Li, Jing Li, Tengxiao Liang, Ping Song, Yongan Ye, Jinsheng Yang, Ning Li, Ruodan Xu
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Macromolecular Bioscience.2019;[Epub] CrossRef
Cell biology of microbes and pharmacology of antimicrobial drugs explored by Atomic Force Microscopy
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Research Support, Non-U.S. Gov'ts

The Role as Inoculum Sources of Xanthomonas citri pv. citri Surviving on the Infected Satsuma mandarin Fruits: So Young Kang , Ki Deok Kim , Jeum Kyu Hong , He Nam Hyun , Yong Chull Jeun; J. Microbiol. 2014;52(5):422-426. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3366-z

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Abstract

Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solu-tion II). It indicated that the pathogenicity of the citrus can-ker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solu-tion directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result supports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.

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Seasonal and post-harvest population dynamics of the Asiatic citrus canker pathogen Xanthomonas citri subsp. citri on grapefruit in Florida
Weiqi Luo, Drew Posny, Alissa B. Kriss, Jim H. Graham, Gavin H. Poole, Earl L. Taylor, Greg McCollum, Tim R. Gottwald, Clive H. Bock
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Effects of Nonionic Surfactants on Xanthan Gum Production: a Survey on Cellular Interactions
Tahereh Ghashghaei, Mohammad Reza Soudi, Saman Hoseinkhani, Morteza Shiri
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NOTE] Effects of Light Intensity on Components and Topographical Structures of Extracellular Polysaccharides from the Cyanobacteria Nostoc sp.: Hongmei Ge , Ling Xia , Xuping Zhou , Delu Zhang , Chunxiang Hu; J. Microbiol. 2014;52(2):179-183. Published online February 1, 2014; DOI: https://doi.org/10.1007/s12275-014-2720-5

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Abstract

A study on the effects of light intensity (40 and 80 μE/m2/sec) on the components and topographical structures of extracellular polysaccharides (EPS) was carried out in cyanobacteria Nostoc sp.. EPS yield increased with light intensity. However, light intensity did not significantly affect the EPS fractions and monosaccharide composition. Higher light intensity generally resulted in higher protein content of EPS in similar fractions. The topographical structure of EPS, investigated by atomic force microscopy, appeared as spherical lumps, chains and networks. The long chains were observed at higher light intensity. Thus, light intensity affected the yield and nature of EPS.

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Live and Dead GFP-Tagged Bacteria Showed Indistinguishable Fluorescence in Caenorhabditis elegans Gut: Ju-Ya Hsiao , Chun-Yao Chen , Mei-Jun Yang , Han-Chen Ho; J. Microbiol. 2013;51(3):367-372. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2589-8

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Abstract: Caenorhabditis elegans has been used for studying hostpathogen interactions since long, and many virulence genes of pathogens have been successfully identified. In several studies, fluorescent pathogens were fed to C. elegans and fluorescence observed in the gut was considered an indicator for bacterial colonization. However, the grinder in the pharynx of these nematodes supposedly crushes the bacterial cells, and the ground material is delivered to the intestine for nutrient absorption. Therefore, it remains unclear whether intact bacteria pass through the grinder and colonize in the intestine. Here we investigated whether the appearance of fluorescence is indicative of intact bacteria in the gut using both fluorescence microscopy and transmission electron microscopy. In wild-type N2 C. elegans, Escherichia coli DH5α, and Vibrio vulnificus 93U204, both of which express the green fluorescence protein, were found intact only proximal to the grinder, while crushed bacterial debris was found in the post-pharyngeal lumen. Nevertheless, the fluorescence was evident throughout the lumen of worm intestines irrespective of whether the bacteria were intact or not. We further investigated the interaction of the bacteria with C. elegans phm-2 mutant, which has a dysfunctional grinder. Both strains of bacteria were found to be intact and accumulated in the pharynx and intestine owing to the defective grinder. The fluorescence intensity of intact bacteria in phm-2 worms was indistinguishable from that of crushed bacterial debris in N2 worms. Therefore, appearance of fluorescence in the C. elegans intestine should not be directly interpreted as successful bacterial colonization in the intestine.

Predicting the Chemical Composition and Structure of Aspergillus nidulans Hyphal Wall Surface by Atomic Force Microscopy: Hyun-uk Lee , Jong Bae Park , Haeseong Lee , Keon-Sang Chae , Dong-Min Han , Kwang-Yeop Jahng; J. Microbiol. 2010;48(2):243-248. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-8094-4

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Abstract: In fungi, cell wall plays an important role in growth and development. Major macromolecular constituents of the aspergilli cell wall are glucan, chitin, and protein. We examined the chemical composition and structure of the Aspergillus nidulans hyphal wall surface by an atomic force microscope (AFM). To determine the composition of the cell wall surface, the adhesion forces of commercially available β-glucan, chitin, and various proteins were compared to those of corresponding fractions prepared from the hyphal wall. In both setups, the adhesion forces of β-glucan, chitin, and protein were 25-50, 1000-3000, and 125-300 nN, respectively. Adhesion force analysis demonstrated that the cell surface of the apical tip region might contain primarily chitin and β-glucan and relatively a little protein. This analysis also showed the chemical composition of the hyphal surface of the mid-region would be different from that of the apical region. Morphological images obtained by the tapping mode of AFM revealed that the hyphal tip surface has moderate roughness.

The Three-Dimensional Morphology of Candida albicans as Seen by High-Resolution Scanning Electron Microscopy: Michela Isola , Raffaella Isola , Maria Serenella Lantini , Alessandro Riva; J. Microbiol. 2009;47(3):260-264. Published online June 26, 2009; DOI: https://doi.org/10.1007/s12275-008-0212-1

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Abstract: The fine structure of Candida albicans has been repeatedly described by transmission electron microscopy, whereas studies by high-resolution scanning electron microscopy (HRSEM) are rare and devoted solely to the study of its external morphology. This report describes the results of an HRSEM study on C. albicans carried out by an osmium maceration protocol modified to better retain the structural characteristics of this yeast. Thus, we visualized various intracellular structures including invaginations of cell membrane (plasmalemmasomes), nuclear envelope, mitochondria, the vacuolar system, and two additional structures that might represent a form of endoplasmic reticulum and the Golgi apparatus. The present investigation, which for the first time shows the organelles of C. albicans at the 3D level, may lead to a better understanding of its cell physiology.

An Examination of the Bacteriophages and Bacteria of the Namib Desert: Eric Prestel , Sylvie Salamitou , Michael S. DuBow; J. Microbiol. 2008;46(4):364-372. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0007-4

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Abstract: Bacteria and their viruses (called bacteriophages, or phages), have been found in virtually every ecological niche on Earth. Arid regions, including their most extreme form called deserts, represent the single largest ecosystem type on the Earth''s terrestrial surface. The Namib desert is believed to be the oldest (80 million years) desert. We report here an initial analysis of bacteriophages isolated from the Namib desert using a combination of electron microscopy and genomic approaches. The virus-like particles observed by electron microscopy revealed 20 seemingly different phage-like morphologies and sizes belonging to the Myoviridae and Siphoviridae families of tailed phages. Pulsed-field gel electrophoresis revealed a majority of phage genomes of 55~65 kb in length, with genomes of approximately 200, 300, and 350 kb also observable. Sample sequencing of cloned phage DNA fragments revealed that approximately 50% appeared to be of bacterial origin. Of the remaining DNA sequences, approximately 50% displayed no significant match to any sequence in the databases. The majority of the 16S rDNA sequences amplified from DNA extracted from the sand displayed considerable (94~98%) homology to members of the Firmicutes, and in particular to members of the genus Bacillus, though members of the Bacteroidetes, Planctomycetes, Chloroflexi, and delta-Proteobacteria groups were also observed.

Antifungal Activities of the Essential Oils in Syzygium aromaticum (L.) Merr. Et Perry and Leptospermum petersonii Bailey and their Constituents against Various Dermatophytes: Mi-Jin Park , Ki-Seob Gwak , In Yang , Won-Sil Choi , Hyun-Jin Jo , Je-Won Chang , Eui-Bae Jeung , In-Gyu Choi; J. Microbiol. 2007;45(5):460-465.; DOI: https://doi.org/2589 [pii]

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Abstract: This study was carried out in order to investigate the potential of using plant oils derived from Leptospermum petersonii Bailey and Syzygium aromaticum L. Merr. Et Perry as natural antifungal agents. The antifungal effects of essential oils at concentrations of 0.05, 0.1, 0.15, and 0.2 mg/ml on the dermatophytes Microsporum canis (KCTC 6591), Trichophyton mentagrophytes (KCTC 6077), Trichophyton rubrum (KCCM 60443), Epidermophyton floccosum (KCCM 11667), and Microsporum gypseum were evaluated using the agar diffusion method. The major constituents of the active fraction against the dermatophytes were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography analysis. The antifungal activities of S. aromaticum oil (clove oil) against the dermatophytes tested were highest at a concentration of 0.2 mg/ml, with an effectiveness of more than 60%. Hyphal growth was completely inhibited in T. mentagrophytes, T. rubrum, and M. gypseum by treatment with clove oil at a concentration of 0.2 mg/ml. Eugenol was the most effective antifungal constituent of clove oil against the dermatophytes T. mentagrophytes and M. canis. Morphological changes in the hyphae of T. mentagrophytes, such as damage to the cell wall and cell membrane and the expansion of the endoplasmic reticulum, after treatment with 0.11 mg/ml eugenol were observed by transmission electron microscopy (TEM). At a concentration of 0.2 mg/ml, L. petersonii oil (LPO) was more than 90% effective against all of the dermatophytes tested, with the exception of T. rubrum. Geranial was determined to be the most active antifungal constituent of L. petersonii oil. Taken together, the results of this study demonstrate that clove and tea tree oils exhibited significant antifungal activities against the dermatophytes tested in this study.

Physiological importance of trypsin-like protease during morphological differentiation of streptomycetes: Kim, In Seop , Kang, Sung Gyun , Lee, Kye Joon; J. Microbiol. 1995;33(4):315-321.

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Abstract: The relationship between morphological differentiation and production of trypsin-like protease (TLP_ in streptomycetes was studied. All the Streptomyces spp. In this study produced TLP just before the onset of aerial mycelium formation. Addition of TLP inhibitor, TLCK, to the top surface of colonies inhibited aerial mycelium formation as well as TLP inhibitor, TLCK, to the top surface of colonies inhibited aerial mycelium formation as well as TLP activity. Addition of 2% glucose to the Bennett agar medium repressed both the aerial mycelium formation and TLP production in S. abuvaviensis, S. coelicolor A3(2), S exfoliatus, S. microflavus, S. roseus, s. lavendulae, and S. rochei. However the addition of glucose did not affect S. limosus, S. felleus, S. griseus, S. phaechromogenes, and S. rimosus. The glucose repression on aerial mycelium formation and production of TLP was relieved by the addition of glucose anti-metabolite (methyl α-glucopyranoside). Therefore, it was concluded that TLP production is coordinately regulated with morphological differentiation and TLP activity is essential for morphological differentiation in streptomycetes. The proposed role of TLP is that TLP participates in the degradation of substrate mycelium protein for providing nutrient for aerial mycelial growth.

Fluorescence Microscopy of Condensed DNA Conformations of Bacterial Cells: Erhan Suleymanoglu; J. Microbiol. 2002;40(4):319-326.

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Abstract: Cellular DNA in prokaryotes is organized in nucleic acid-protein self-assemblies referred to as the nucleoid. The physical forces responsible for its stability inside the poor solvent properties of the cytoplasm and their functional implications are not understood. Studies on the organisation and functioning of the cytosol of cells largely rely on experimental protocols performed in highly dilute solutions using biochemically purified molecules, which is not a reliable substitute for the situation existing in vivo. Our current research interest is focused on the characterization of biological and physical forces determining the compaction and phase separation of DNA in Escherichia coli cytoplasm. We have emphasized the effect of excluded volume in solutions with high macromolecular concentrations (macromolecular crowding) upon self-association patterns of reactions. The prokaryotic cytosol was simulated by addition of inert polymer polyethylene glycol (PEG) (average molecular weight 20000), as an agent which afterwards facilitates the self-association of macromolecules. Fluorescence microscopy was used for direct visualization of nucleoids in intact cells, after staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride). Addition of the crowding agent PEG 20,000, in increasing concentrations generated progressively enhanced nucleoid compaction, the effect being stronger in the presence of 0.2 M NaCl and 5 uM MgCl_2. Under these conditions, the nucleoids were compacted to volumes of around 2㎛^3 or comparable sizes with that of living cells.

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