Bacterial Colony Morphology Research Articles

A specific innate immune response silences the virulence of Pseudomonas aeruginosa in a latent infection model in the Drosophila melanogaster host.

Microbial pathogenicity often depends on the route of infection. For instance, P. aeruginosa or S. marcescens cause acute systemic infections when low numbers of bacteria are injected into D. melanogaster flies whereas flies succumb much slower to the continuous ingestion of these pathogens, even though both manage to escape from the gut compartment and reach the hemocoel. Here, we have developed a latent P. aeruginosa infection model by feeding flies on the bacteria for a short period. The bacteria stably colonize internal tissues yet hardly cause any damage since latently-infected flies live almost as long as noninfected control flies. The apparently dormant bacteria display particular characteristics in terms of bacterial colony morphology, composition of the outer cell wall, and motility. The virulence of these bacteria can however be reactivated upon wounding the host. We show that melanization but not the cellular or the systemic humoral response is the predominant host defense that establishes latency and may coerce the bacteria to a dormant state. In addition, the lasting activation of the melanization responses in latently-infected flies provides a degree of protection to the host against a secondary fungal infection. Latent infection by an ingested pathogen protects against a variety of homologous or heterologous systemic secondary infectious challenges, a situation previously described for the endosymbiotic Wolbachia bacteria, a guard against viral infections.

Early Classification of Gram-negative Bacteria with Colony Imaging and Deep Learning without Coding Experience

Bacterial colony morphology is the first step in classifying bacterial species during the microbial identification process. It is very important to assess the morphology of bacterial colonies in a preliminary screening process to largely reduce the scope of possible bacteria species and increase work productivity in clinical bacteriology by making later identification more specific. However, making a decision about this topic requires sufficient clinical laboratory expertise. Teachable Machine® is a rapid, easy-to-use, web-based tool accessible to everyone that is used to create machine learning models. In this study, the performance of Teachable Machine® was assessed for cheap, rapid and practical identification of enteric and non-fermenting bacteria frequently isolated in microbiology laboratories. A total of 1202 colony images were used to train and validate the network's diagnostic performance. Additionally, 80 representative test images were used to assess performance. Level 1 was defined as E. coli-K. pneumonia, Level 2 was defined as P. aeruginosa-A. baumannii, Level 3 was defined as enteric bacteria-non-fermenting bacteria and Level 4 was defined as differentiating these four pathogens from each other. Mean accuracy of Teachable Machine® for the defined classes was 96.7%, 94.1%, 94.3%, and 90.3% for Levels 1, 2, 3, and 4, respectively. General accuracy for classification of the 80 representative colonies was 82.5% and the hit rates were 85.0%, 100%, 75.0%, and 70.0% for E. coli, K. pneumoniae, P. aeruginosa and A. baumannii, respectively. This cost-effective bacterial identification system, supported by deep learning, will be an important pioneer for a variety of applications in clinical microbiology by reducing the identification process by a significant degree and automating identification of colonies without requiring a specialist.

SELECTION OF ANTAGONISTIC RHIZOBACTERIA POTENTIAL FOR BIOLOGICAL CONTROL OF Fusarium oxysporum

Fusarium oxysporum is a pathogen that causes wilt disease in many plants and can couse losses of up to fifty percent. Standard control is to use synthetic fungicides. However, continuous use of fungicides has a high environmental risk, so environmentally friendly and safe control efforts are needed. One way is by using rhizosphere bacteria or rhizobacteria. This research aims to obtain rhizobacteria isolates that can control the pathogen F. oxysporum, which causes fusarium wilt in vitro. This research was conducted from June to July 2023. The methods in this research included isolating rhizobacteria, observing the morphology of bacterial colonies, gram staining, gram reaction test, catalyst test, and in vitro antagonist test. Research data shows that of the thirty rhizobacteria isolates observed, all isolates had the potential to inhibit the growth of F. oxysporum. The KMTK2 showed the highest inhibitory ability isolate with an inhibitory ability of 73,99%, and the lowest inhibitory ability was shown by the TBA1 isolate with an inhibitory ability of 51,56%. Potential rhizobacteria isolates can suppress the growth of the pathogen F. oxysporum and can be used as a biological agent to reduce the use of pesticides in treating plant diseases.

Antagonistic Test of Bacteria Producing Siderophore and Protease Enzymes from The Rhizosfer of Peanut Plants on The Growth of Pathogenic Fungus Colletotrichum gloeosporioides

This study aims to determine the inhibitory effect of bacterial isolates producing siderophores and protease enzymes on the growth of the pathogenic fungus Colletotrichum gloeosporioides. The initial stage of research begins with the isolation of pathogenic bacteria and fungi, and is followed by testing the production of siderophores and protease enzymes. Bacteria were isolated from the rhizosphere of peanut plants, while pathogenic fungi were isolated from large chili fruit infected with anthracnose disease taken from Jelantik Village, Central Lombok Regency. Characterization of isolates for siderophore production used the Arnow method, while the protease enzyme production test used SKIM Milk Agar media. Next, the inhibition test of bacterial isolates against pathogenic fungi was carried out using the dual culture method. Characterization of potential isolates was carried out by observing bacterial colony morphology, gram staining and biochemical tests. The results of the siderophore production test showed that there was one isolate capable of producing siderophores with the isolate code RKT2. Meanwhile, the protease enzyme production test showed that all bacterial isolates produced protease enzymes, where isolate RKT9 had the highest Proteolytic Index, namely 1.57. The two isolates showed different inhibitory test results, namely isolate RKT2 had high inhibition, while RKT9 had low inhibition. The results of the research showed that a bacterial isolate (RKT2) from the rhizosphere of peanut plants was able to inhibit the growth of the pathogenic fungus Colletotrichum gloeosporioides in the high category.

ISOLASI DAN KARAKTERISASI BAKTERI AMILOLITIK DARI SAMPEL AIR PANTAI PARIS TIGARAS

Amylolytic bacteria have great genetic diversity with high enzymatic activity and have the expertise to break down starch into simpler materials and degrade organic substances in water. This study aims to determine the variety of types of amylolytic bacteria obtained and to see how the characterization of bacteria is isolated from the waters of Paris Tigaras Beach. Isolation of amylolytic bacteria from the waters of Paris Tigaras Beach was carried out using the spread plate method taken from the last dilution of 10<sup>-5</sup>. Isolates ST3 03 and ST3 04 showed amylolytic activity by showing a clear zone of 11.8 mm in isolates ST3 03 and 8.2 mm in isolates ST3 04. Characterization of isolates ST3 03 and ST3 04 was observed including Gram-positive bacteria with rod-shaped cells, that can produce catalase enzymes, ferment three types of sugars, and move (motil). From the results of observations of the morphology of bacterial colonies and cells, and biochemical tests, isolates of amylolytic bacteria ST3 03 and ST3 04 belong to the genus Bacillus.

Morphology and Size of Bacterial Colonies Control Anoxic Microenvironment Formation in Porous Media.

Bacterial metabolisms using electron acceptors other than oxygen (e.g., methanogenesis and fermentation) largely contribute to element cycling and natural contaminant attenuation/mobilization, even in well-oxygenated porous environments, such as shallow aquifers. This paradox is commonly explained by the occurrence of small-scale anoxic microenvironments generated by the coupling of bacterial respiration and dissolved oxygen (O2) transport by pore water. Such microenvironments allow facultative anaerobic bacteria to proliferate in oxic environments. Microenvironment dynamics are still poorly understood due to the challenge of directly observing biomass and O2 distributions at the microscale within an opaque sediment matrix. To overcome these limitations, we integrated a microfluidic device with transparent O2 planar optical sensors to measure the temporal behavior of dissolved O2 concentrations and biomass distributions with time-lapse videomicroscopy. Our results reveal that bacterial colony morphology, which is highly variable in flowing porous systems, controls the formation of anoxic microenvironments. We rationalize our observations through a colony-scale Damköhler number comparing dissolved O2 diffusion and a bacterial O2 uptake rate. Our Damköhler number enables us to predict the pore space fraction occupied by anoxic microenvironments in our system for a given bacterial organization.

Confirmation of Burkholderia gladioli as the Causal Agent of Bacterial Scab on Gladiolus (Gladiolus spp.) in Ohio.

Ohio is one of the top five floriculture producers in the United States, grossing over $200 million annually (NASS 2019). Within the international floriculture trade, gladiolus cut flowers represent the fifth highest grossing crop (Ahmed et al. 2002). In September 2021, the Ornamental Crops Pathology Lab at the Ohio State University received a gladiolus (Gladiolus spp.) sample of an unknown cultivar from a home garden in Franklin Co., OH where several plants had failed to grow from planted corms or were stunted and displaying symptoms of disease. Bleached, water-soaked spots with necrotic margins along the flowering stems, stunted flowers with partial necrosis, and necrotic bracts were observed on the submitted sample. Bacterial isolations were performed by surface disinfesting small sections of bract tissue from the border of a lesion by soaking in 10% bleach for 30 sec and rinsing twice in sterile water, macerating the tissue in sterile water, and streaking the suspension on nutrient agar (NA) plates. Plates were incubated at 28°C for 48 hours and the resulting colonies were purified by re-streaking a single colony on NA twice. Bacterial colony morphology on NA presented as cream-colored and shiny with an irregular form and undulate margin. Five in vitro tests were performed using one representative isolate to identify the bacterium to the genus level: (1) confirmed levan production, (2) confirmed pectinolytic activity, (3) confirmed ability to grow at 40°C, (4) inability to grow under anaerobic conditions, and (5) a negative oxidase test (Schaad et al. 2000). All test results identified the genus as Burkholderia. To identify to species level, gyrase subunit B (gyrB) and RNA polymerase subunit D (rpoD) markers were PCR amplified and sequenced using primers UP1-E/AprU, and 70F2/70R2, respectively (Maeda et al. 2006). NCBI GenBank BLASTn comparison showed that the gyrB sequence shared 99.33% identity to the type strain of B. gladioli (CP009323.1), while the rpoD sequence showed 99.53% identity (CP009322.1). Sequences were deposited in GenBank under accession numbers ON597852 (gyrB) and ON597853 (rpoD). To confirm pathogenicity, each of two Gladiolus communis 'Mini Elvira' potted plants were inoculated with two bacterial and two control treatments (3 leaves/treatment/plant) as follows: leaf infiltration with 1 mL of either (i) a distilled water-Tween 20 (0.03% v/v) bacterial suspension (106 cfu/mL) or (ii) a sterile water-Tween 20 suspension using a needle-less syringe; foliar spray with either (iii) the bacterial suspension or (iv) water-Tween suspension until run-off. Following inoculation, plants were covered for 24 hours with a plastic bag to increase humidity and favor infection and maintained in a greenhouse at an average temperature of 23°C. After 3 days, water-soaked, necrotic lesions were observed on the inoculated plants regardless of inoculation method, while control leaves remained asymptomatic. To fulfill Koch's postulates, bacteria were re-isolated from the lesions 7 days post-inoculation and confirmed to be identical to the original isolate based on rpoD gene sequencing. Bacterial scab of gladiolus was reported in Ohio in the late 1900s as caused by Pseudomonas gladioli (syn. P. marginata; Ellett, 1989). To the best of our knowledge, this report represents the first molecular identification of the causal agent as Burkholderia gladioli. In Ohio, the pathogen has also been observed causing slippery skin on onion but not officially reported in the peer-reviewed literature. Additionally, B. gladioli has been reported in other parts of the United States on orchid, corn, and rice (Keith et al. 2005; Lu et al. 2007; Nandakumar et al. 2009). Given the significant role of gladiolus within Ohio's floricultural trade, as well as the ability of this pathogen to infect other regional crops, monitoring of bacterial scab is important for floriculture and field crop growers alike.

Characterization and Antagonistic Effect of Culturable Apple-Phyllosphere Endophytic Bacteria from the Cold Plateau in Yunnan, China

The endophytic bacteria in apple leaves from apple-producing areas of Yunnan, China were isolated and identified on the basis of bacterial colony morphology and nucleotide sequences of 16S rRNA and rpoB genes. The endophytic bacterial isolates with nitrogen, phosphorus, and potassium utilization abilities were screened by culturing on functional media. A total of 5709 isolates of culturable endophytic bacteria (CEB) were isolated from 30 apple leaf samples collected from different regions. A total of 39 CEB representative isolates were identified as Bacillus velezensis, B. subtilis, B. licheniformis, B. safensis, B. pumilus, and Priestia megaterium. Among them, B. velezensis and B. subtilis were the main CEB, accounting for 55.00% and 34.37%, respectively, which exhibited potential inhibition on not only the main apple disease pathogens of Alternaria alternata, Valsa mali, Fusarium oxysporum, and Rosellinia necatrix, but also some important and uncontrollable phytopathogens, including F. oxysporum f. sp. cubense that causes banana Fusarium wilt, and Phytophthora nicotianae that causes tobacco black shank. Among these isolated endophytic bacteria species, a total of 10 strains, including b3, b4, b16, b17, b20, and b23 of B. subtilis, b7, b24, and b28 of B. licheniformis, and b38 of B. velezensis, can fix nitrogen; 8 strains, including b7 and b28 of B. licheniformis, b5, b10, and b23 of B. subtilis, b8 of B. safensis, and b6 of Priestia megaterium, could dissolve inorganic phosphorus; 11 strains, including b9, b12, b14, b30, b34, and b43 of B. velezensis, b6 of Priestia megaterium, and b17, b18, b20, and b26 of B. subtilis, could degrade organic phosphorus; and 5 strains, including b4, b5, and b26 of B. subtilis, and b7 and b28 of B. licheniformis could dissolve potassium. These strains are valuable resources of endophytic bacteria that have adapted to the ecological environment of the Cold Plateau apple-production area and could be used as plant disease biocontrol agents and biofertilizers of crops. The culturable phyllosphere endophytes in apple leaves relate to geographic locations, apple varieties, and environment, providing the basis to explain the mechanisms underlying the establishment of apple endophyte diversity and may help to devise apple disease management strategies.

Molecular identification, whole-cell protein profiling, antibiotics sensitivity and pathogenicity of Chryseobacterium gleum recovered from diseased fishes of freshwater wetlands in India

Globally, bacterial gill disease is one of the most prominent bacterial diseases affecting both marine and freshwater fishes. This study aimed to investigate diseased Labeo rohita, Labeo catla and Oreochromis niloticus with gill discoloration, tail rot and skin ulcer that resembles the typical sign of bacterial gill disease from East Kolkata Wetlands, India. The results revealed that four isolates of Chryseobacterium gleum were the responsible pathogens. The isolates CIFRI-RRM3, CIFRI-BFG2, CIFRI-SRM4 and CIFRI-TRM5 were identified using bacterial colony morphology, biochemical test, whole-cell protein profiling, 16S rRNA gene amplification and nucleotide sequence analysis. The antibiotic susceptibility assay showed that all the isolates were completely resistant to ampicillin, amoxyclav, cefalexin, cephalothin, ceftriaxone, ceftazidime, cefotaxime, cefuroxime, cefadroxil, chloramphenicol, kanamycin, netilmicin, penicillin-G, ticarcillin, tobramycin, erythromycin and tetracycline. A challenge experiment was also conducted for four isolates with intraperitoneal injection at a dose of 2.5 × 1010 CFU mL−1 to 4.2 × 1010 CFU mL−1in respective fish hosts. Only three isolates (CIFRI-BFG2, CIFRI-RRM3 and CIFRI-TRM5) proved their virulence by producing diseases with mild symptoms of skin lesions, gill disease and cumulative mortality of 10–20%. To the best of our knowledge, this is the first description of the phenotypic, molecular and whole-cell protein profiling of C. gleum derived from freshwater fishes in India.

Isolation of Entomopathogenic Lysinibacillus sphaericus from Sewage at Some Housing Complex in Mataram City and Evaluation of Its Toxicity Against Aedes aegypti Larvae in Laboratory

The aims of this study were to isolate Lysinibacillus sphaericus from sewage at some housing complex in Mataram city and evaluate its toxicity against third Aedes aegypti larvae. The bacteria collected from mud taken from diluted sewage and objected to heat shock procedure at 80ºC for 30 minutes. The microbiological culture was done using a Nutrient Agar solid medium and incubated at 30ºC for 72 hours. Bacterial characterization was done based on bacterial colony morphology, cell morphology, cell physiology, and cell biochemistry characteristics. Toxicity test on 3rd Aedes aegypti larvae was done for 24, 48, and 72 hours applying Nutrient Broth medium with various dilution. From this study total of 11 isolates of Lysinibacillus sphaericus were isolated, only three isolates showed a high killing rate against Ae. aegypti in 24-hour observation. They were isolate Bs9-2-3 (LC50 24-hour value was 1.75 x 104 cell/mL), isolate Bs9-1-5 (LC50 24-hour value was 6.23 x 104 cell/mL) and isolate Bs2-1-2 (LC50 24-hour value was 7.17 x 106 cell/mL). These local isolates of L. sphaericus had good potential to be developed for bacterial-based biopesticide/biolarvicide for battling Aedes mosquito larvae in the near future.

ANTITUBERCULOSIS EFFECTIVENESS OF ENDOPHYTIC ANDALAS MUTANT BACTERIA PRODUCS INDUCED BY UV LIGHT WILL BE CONDUCTED

Indonesia is the 3rd largest country in the world in cases of tuberculosis (TB) infection. Infections caused by the bacteria Mycobacterium Tuberculosis have claimed 100,000 lives per year. This death rate is expected to continue to increase due to resistance to Antituberculosis Drugs (OAT). One strategy to overcome the increasing number of resistance of M. tuberculosis bacteria to OAT is to find new and better antituberculosis active compounds. This study aims to determine the potential of Andalas Endophytic Bacteria mutated by UV light on antituberculosis activity. This research is descriptive research. The research was conducted at the Research Laboratory of the Faculty of Mathematics and Natural Sciences, Padang State University. The mutation process is carried out as a form of increasing the production of active compounds. Natural anti microbial compounds can be obtained through the fermentation process. The active compounds from the fermentation products were extracted using methanol and aquadest as solvents. Testing against M. tuberculosis using the Mycobacterium Growth Indicator Tube (MGIT) 960 System as a medium for sensitivity testing. The fermented supernatant with absolute concentration and extract of each solvent with a concentration of 75% were mixed into the MGIT 960 tube. The results showed that the length of time UV irradiation could reduce the number of endophytic bacterial cells of Andalas isolate ATB 10-6. UV light also causes changes in the morphology of mutant bacterial colonies. UV irradiation can produce mutant isolates that have better antifungal activity, where of the 8 mutant bacteria produced, 7 mutants have better antifungal activity than the wild type, and the growth of mutant bacteria is faster than the wild type.

Evaluation of use of RPMI medium to preserve cell morphology for pleural/peritoneal fluid cytology

Introduction:Cytologic evaluation is the best way to detect the presence of malignancy in body cavity fluids. Roswell Park Memorial Institute (RPMI) medium or RPMI 1640 is used in cell culture, tissue culture, and also to improve the cellularity and morphology of CSF cytology specimens.Objectives:To determine whether RPMI medium can be used to preserve cell morphology in pleural/peritoneal effusion samples.Method and Material:The study was conducted on 30 pleural/peritoneal fluid samples received routinely during 2 months for diagnostic purposes in our department. The samples were divided into four parts. One-fourth of the sample was directly refrigerated and the other fourth was at room temperature. In the other two parts, an equal volume of RPMI media was added, and one was kept at room temperature and the other refrigerated. These cytospin-prepared Giemsa-stained smears were examined for cell morphology, cellularity, and occurrence of bacterial colonies at 24 h, 48 h, 72 h, and 1 week, respectively.Result:Refrigerated RPMI medium is the best preservative for pleural/peritoneal samples; however, samples with RPMI at room temperature were equivalent/even worse than the simple refrigerated sample.

Association of Oral Microbiota Properties on Hyaluronidase and Biofilm Formation

Correlation between hyaluronidase (Hyl) activity and biofilm detachment in a few bacterial species was found. However, it is unclear if this association applies to bacterial species or for more general bacterial characteristics. This study determined the association between biofilm production and Hyl activity among bacterial isolates from the oral cavity of healthy subjects, and its association with Gram staining group, colony surface morphology and bacteria shape. The swab was taken from the tongue, cheek and entire teeth surfaces of 35 subjects, and tested for biofilm through modified microtiter plate assay while Hyl production was screened through HA rapid plate method. Forty-four isolates were found, each 50% are Gram-positive, and Gram-negative bacteria, with the majority are cocci and non-mucoid colony. More than 70% of isolates are moderate and strong; (n= 17, 38.6%) and (n=15, 34.1%) respectively for biofilm production; and 68.2% are Hyl producer. A significant association was found between Hyl and bacterial shape (p=0.018) and colony morphology (p=0.018), while other association is not significantly measured, including between Hyl and biofilm (p=0.659). This study showed that biofilm production is not affected by the characteristics of the bacteria to produce or not produce hyaluronidase. Meanwhile, Hyl production is prone in rod shape and mucoid isolates which need further investigations.

Subunit vaccine protects against a clinical isolate of Mycobacterium avium in wild type and immunocompromised mouse models

The nontuberculous mycobacteria (NTM) Mycobacterium avium is a clinically significant pathogen that can cause a wide range of maladies, including tuberculosis-like pulmonary disease. An immunocompromised host status, either genetically or acutely acquired, presents a large risk for progressive NTM infections. Due to this quietly emerging health threat, we evaluated the ability of a recombinant fusion protein ID91 combined with GLA-SE [glucopyranosyl lipid adjuvant, a toll like receptor 4 agonist formulated in an oil-in-water stable nano-emulsion] to confer protection in both C57BL/6 (wild type) and Beige (immunocompromised) mouse models. We optimized an aerosol challenge model using a clinical NTM isolate: M. avium 2-151 smt, observed bacterial growth kinetics, colony morphology, drug sensitivity and histopathology, characterized the influx of pulmonary immune cells, and confirmed the immunogenicity of ID91 in both mouse models. To determine prophylactic vaccine efficacy against this M. avium isolate, mice were immunized with either ID91 + GLA-SE or bacillus Calmette–Guérin (BCG). Immunocompromised Beige mice displayed a delayed influx of innate and adaptive immune cells resulting in a sustained and increased bacterial burden in the lungs and spleen compared to C57BL/6 mice. Importantly, both ID91 + GLA-SE and BCG vaccines significantly reduced pulmonary bacterial burden in both mouse strains. This work is a proof-of-concept study of subunit vaccine-induced protection against NTM.

Mycobacterium tuberculosis PPE10 (Rv0442c) alters host cell apoptosis and cytokine profile via linear ubiquitin chain assembly complex HOIP-NF-κB signaling axis

Tuberculosis caused by Mycobacterium tuberculosis infection remains one of the top ten causes of deaths worldwide. M. tuberculosis genome devoted 10% capacity for highly repeated PE/PPE genes family. To explore the role of PPE10 in host-pathogen interaction, PPE10 encoding gene Rv0442c was heterologously expressed in the nonpathogenic M. smegmatis strain. PPE10 altered the bacterial cell surface properties, colony morphology, and biofilm formation. Ms_PPE10 showed more resistance to stress conditions such as diamide, and low pH, as well as higher survival within the macrophage. Moreover, the host's cell apoptosis was regulated via decreased expression of caspases, IL-1, IL-6, and TNF-α through the Linear Ubiquitin Chain Assembly Complex (LUBAC) HOIP-NF-κB signaling axis. The study revealed novel insights into the mechanism of action of the PPE family.

Isolation and Characterization of Bacteria from River Origin in Mandailing Natal North Sumatera

The indicator of water pollution according to microbiological standards is the presence of microbiological pollutants. To see the presence of bacteria in the waters, it is necessary to study the isolation and characterization of these bacteria. The existence of the river in the Mandailing Natal area has experienced a change in character due to gold mining activities along the river flow. The purpose of this study was to determine the types of bacteria in the river in the Mandailing Natal area of North Sumatra. This research was conducted at the Laboratory of Microbiology, Department of Biology, Faculty of Mathematics and Natural Sciences, State University of Medan. The research period was from June to October 2020. The population in this study was the water of the Batang Gadis Mandailing Natal river, North Sumatra. The sample in this study is the Batang Gadis river water around the Mandailing Natal gold mine, North Sumatra. The research was carried out by taking samples and isolating bacteria which included planting the samples on media enriching the planting of the samples on various growing media, purifying bacterial colonies and observing the morphology of bacterial colonies. The results showed that there were 6 bacterial isolates living in river waters in Mandailing Natal, North Sumatra.

Structural Characterization of Functional Ingredient Levan Synthesized by Bacillus siamensis Isolated from Traditional Fermented Food in Thailand

The rising global population continues to threaten the world’s food security. The discovery of new technologies to produce food of nutritional and functional properties is urgently needed. One beneficial food to humans of known nutritional value is the prebiotic levan. To address the problem, the present work is aimed at isolating levansucrase enzyme-producing microorganisms from traditional fermented food in Thailand. Bacterial colony morphology was observed for mucoidal consistency on culture plates. Isolated colonies were characterized morphologically by gram staining methods. Dinitrosalicylic acid (DNS) and thin-layer chromatography (TLC) reported the highest microbial enzyme activity of 8.51 IU/ml at 12 hours via hydrolysis and frutotransferase activities. Structural characterization of levan via Fourier-transform infrared spectroscopy (FTIR) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy showed β-(2,6)-fructofuranose linkages. The highest enzyme activity was exhibited by bacterium B-6 identified as Bacillus siamensis NR 11274.1 based on the 16s rDNA gene sequence analyses. Thus, the isolated bacterium from the traditional food was confirmed to produce levansucrase enzyme of high industrial importance for the synthesis of levan as a functional food.

Regulation of Exopolysaccharide Production by ProE, a Cyclic-Di-GMP Phosphodiesterase in Pseudomonas aeruginosa PAO1.

The ubiquitous second messenger c-di-GMP is involved in regulation of multiple biological functions including the important extracellular matrix exopolysaccharides (EPS). But how c-di-GMP metabolic proteins influence EPS and their enzymatic properties are not fully understood. Here we showed that deletion of proE, which encodes a protein with GGDEF-EAL hybrid domains, significantly increased the transcriptional expression of the genes encoding EPS production in Pseudomonas aeruginosa PAO1 and changed the bacterial colony morphology. Our data showed that ProE is a very active phosphodiesterase (PDE), with a high enzyme activity in degradation of c-di-GMP. Interestingly, the optimal activity of ProE was found in the presence of Co2+, unlike other PDEs that commonly rely on Mg2+ or Mn2+ for best performance. Furthermore, we identified three widely conserved novel residues that are critical for the function of ProE through site-directed mutagenesis. Subsequent study showed that ProE, together with other three key PDEs, i.e., RbdA, BifA, and DipA regulate the EPS production in P. aeruginosa PAO1. Moreover, by using the GFP-fusion approach, we observed that these four EPS associated-PDEs showed a polar localization pattern in general. Taken together, our data unveil the molecular mechanisms of ProE in regulation of EPS production, and provide a new insight on its enzymatic properties in degradation of c-di-GMP.

Inside Cover: Generalized spectral light scatter models of diverse bacterial colony morphologies (J. Biophotonics 12/2019)

The forward elastic‐light‐scattering pattern of a bacterial colony reflects its morphological characteristics. Three bacteria genera whose colonies having convex, crateriform, or irregular elevation were investigated to study the correlation between the morphology and the scattering pattern of the colony. The difference in the colony elevation produced distinct shapes of light diffraction in the scattering pattern, resulting circular diffraction rings or scattered light.Further details can be found in the article by Iyll‐Joon Doh, Jennifer Sturgis, Diana V. Sarria Zuniga, et al. (e201900149). image

Exploration of the effects of a degS mutant on the growth of Vibrio cholerae and the global regulatory function of degS by RNA sequencing.

BackgroundDegS is a periplasmic serine protease that is considered to be the initiator of the σE stress response pathway, and this protein plays an important role in the regulation of the stress response in E. coli. However, knowledge of the biological function and global regulatory network of DegS in Vibrio cholerae remains limited. In this study, we aimed to characterize the molecular functions and further investigate the regulatory network of degS in V. cholerae.MethodsA deletion mutant of degS was constructed in the V. cholerae HN375 strain. Bacterial colony morphology was observed by a plate-based growth experiment, and bacterial growth ability was observed by a growth curve experiment. High-throughput RNA sequencing (RNA-Seq) technology was used to analyze the differential transcriptomic profiles between the wild-type and degS mutant strains. Gene ontology (GO), pathway analysis and Gene-Act-network analysis were performed to explore the main functions of the differentially expressed genes. Quantitative real-time PCR (qRT-PCR) was performed to validate the reliability and accuracy of the RNA-Seq analysis. The complementation experiments were used to test the roles of degS and ropS in the small colony degS mutant phenotype.ResultsWhen degS was deleted, the degS mutant exhibited smaller colonies on various media and slower growth than the wild-type strain. A total of 423 differentially expressed genes were identified, including 187 genes that were upregulated in the degS mutant compared to the wild-type strain and 236 genes that were relatively downregulated. GO categories and pathway analysis showed that many differentially expressed genes were associated with various cellular metabolic pathways and the cell cycle. Furthermore, Gene-Act network analysis showed that many differentially expressed genes were involved in cellular metabolic pathways and bacterial chemotaxis. The cAMP-CRP-RpoS signaling pathway and the LuxPQ signal transduction system were also affected by the degS mutant. The expression patterns of nine randomly selected differentially expressed genes were consistent between the qRT-PCR and RNA-seq results. The complementation experiments showed that the small colony degS mutant phenotype could be partially restored by complementation with the pBAD24-degS or pBAD24-rpoS plasmid.DiscussionThese results suggest that the degS gene is important for normal growth of V. cholerae. Some of the differentially expressed genes were involved in various cellular metabolic processes and the cell cycle, which may be associated with bacterial growth. Several new degS-related regulatory networks were identified. In addition, our results suggested that the cAMP-CRP-RpoS signaling pathway may be involved in the small colony degS mutant phenotype. Overall, we believe that these transcriptomic data will serve as useful genetic resources for research on the functions of degS in V. cholerae.