Pradimicin U, a promising antimicrobial agent isolated from a newly found Nonomuraea composti sp. nov.

Pradimicin U is a new dihydrobenzo[a]naphthacenequinone compound found to be active on a screen designed to investigate compounds with antimicrobial activity, produced by the actinomycete designated strain FMUSA5-5T. The strain was isolated from a bio-fertilizer of Musa spp. collected from Suphanburi province, Thailand. The chemotaxonomic characteristics and 16S rRNA gene analysis revealed that strain FMUSA5-5T is a member of the genus Nonomuraea. Low genome-based taxonomic criteria, average nucleotide identity (ANI) (82.8-88.3%), average amino-acid identity (AAI) (79.4-87.3%), and digital DNA-DNA hybridization (dDDH) (29.5-38.5%) values and several phenotypic differences between strain FMUSA5-5T and its closest type strains of the genus Nonomuraea indicated that strain FMUSA5-5T represents a novel species of the genus Nonomuraea and the name Nonomuraea composti sp. nov. is proposed for the strain. The crude extract from the culture broth of strain FMUSA5-5T displayed promising antimicrobial activity against several pathogens and led to the isolation of a novel secondary metabolite, pradimicin U. Interestingly, this compound displayed a broad spectrum of biological activities such as antimalarial activity against Plasmodium falciparum K1 (IC50 value = 3.65 µg/mL), anti-Mycobacterium tuberculosis H37Ra (MIC value = 25.0 µg/mL), anti-Alternaria brassicicola BCC 42724 (MIC value = 25.0 µg/mL), anti-Bacillus cereus ATCC 11778 and anti-Staphylococcus aureus ATCC 29213 (MIC values = 6.25 and 1.56 µg/mL, respectively). Moreover, the compound possessed strong anti-human small cell lung cancer (NCI-H187) activity with IC50 value of 5.69 µg/mL, while cytotoxicity against human breast cancer (MCF-7) and Vero cells was very weak (IC50 values of 52.49 and 21.84 µg/mL, respectively).

Changpingibacter yushuensis gen. nov., sp. nov., isolated from fluvial sediment in Qinghai Tibet Plateau of China.

Two facultatively anaerobic, short rod-shaped, non-motile, Gram-stain-positive, unknown bacterial strains (JY-X040T and JY-X174) were isolated from fluvial sediments of Tongtian River in Yushu Tibetan Autonomous Prefecture, Qinghai province, China. Cells formed translucent, gray, round and convex colonies, with a diameter of less than 0.5 mm after 5 days of incubation at 30°C on brain heart infusion-5% sheep blood agar. The 16S rRNA gene sequence similarity between strain JY-X040T and Fudania jinshanensis 313T is 93.87%. In the four phylogenetic trees constructed based on the 16S rRNA gene and 423 core genes, the two isolates form an independent branch, phylogenetically closest to F. jinshanensis 313T, but could not be classified as a member of the genus Fudania or any other genus of the family Arcanobacteriaceae. The DNA G + C content of strain JY-X040T was 57.8%. Calculation results of average nucleotide identity, digital DNA-DNA hybridization value and amino acid identity between strain JY-X040T and F. jinshanensis 313T are 69.9%, 22.9%, and 64.1%. The major cellular fatty acids were C16:0 (23%) and C18:1ω9c (22%). The cell-wall peptidoglycan type was A5α (L-Lys-L-Ala-L-Lys-D-Glu). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and four unidentified components. The whole-cell sugars contained rhamnose and ribose. MK-10(H4) was the sole respiratory quinone. The minimum inhibitory concentration of streptomycin was 32 µg/ml. All physiological, biochemical, chemotaxonomic and genomic characteristics support that strains JY-X040T and JY-X174 represent members of a novel species in a new genus, Changpingibacter yushuensis gen. nov., sp. nov. The type strain is JY-X040T (GDMCC 1.1996T = KCTC 49514T).

Nonomuraea cypriaca sp. nov., isolated from soil.

A novel actinobacterium, designated strain K274T, was isolated from soil collected from Zafer Cape (Cape Apostolos Andreas), the easternmost tip of Cyprus on the Karpas peninsula, Magusa, Northern Cyprus, and a polyphasic approach was used for characterization of the strain. The isolate was found to have chemotaxonomic and morphological properties associated with members of the genus Nonomuraea. The strain has the highest similarity to Nonomuraea zeae DSM 100528T with 99.1% similarity value. In the phylogenetic dendogram based on 16S rRNA gene sequence, strain K274T was formed a distinct clade together N. zeae DSM 100528T, 'Nonomuraea basaltis' 160415 (98.9% similarity), and 'Nonomuraea lycopersici' NEAU-DE8(1) (98.2% similarity). The genome sequence of strain K274T was 11.5 Mbp in size with a total of 11,848 protein-coding genes and 75 RNA genes. The genomic G + C content of the novel strain was 69.7 mol%. Both average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) results between the strain and phlyogenetic neighbours were well below the threshold value, and the novelty are supported by phenotypic and chemotaxonomic differences. Because of all these, strain K274T represents a novel species in the genus Nonomuraea, for which the name Nonomuraea cypriaca sp. nov. is proposed. The type strain is K274T (= DSM 45718T = KCTC 29095T).

Comparison of culture-independent and dependent approaches for identification of native arsenic-resistant bacteria and their potential use for arsenic bioremediation.

Arsenic is a common contaminant in gold mine soil and tailings. Microbes present an opportunity for bio-treatment of arsenic, since it is a sustainable and cost-effective approach to remove arsenic from water. However, the development of existing bio-treatment approaches depends on isolation of arsenic-resistant microbes from arsenic contaminated samples. Microbial cultures are commonly used in bio-treatment; however, it is not established whether the structure of the cultured isolates resembles the native microbial community from arsenic-contaminated soil. In this milieu, a culture-independent approach using Illumina sequencing technology was used to profile the microbial community in situ. This was coupled with a culture-dependent technique, that is, isolation using two different growth media, to analyse the microbial population in arsenic laden tailing dam sludge based on the culture-independent sequencing approach, 4 phyla and 8 genera were identified in a sample from the arsenic-rich gold mine. Firmicutes (92.23%) was the dominant phylum, followed by Proteobacteria (3.21%), Actinobacteria (2.41%), and Bacteroidetes (1.49%). The identified genera included Staphylococcus (89.8%), Pseudomonas (1.25), Corynebacterium (0.82), Prevotella (0.54%), Megamonas (0.38%) and Sphingomonas (0.36%). The Shannon index value (3.05) and Simpson index value (0.1661) indicated low diversity in arsenic laden tailing. The culture dependent method exposed significant similarities with culture independent methods at the phylum level with Firmicutes, Proteobacteria and Actinobacteria, being common, and Firmicutes was the dominant phylum whereas, at the genus level, only Pseudomonas was presented by both methods. It showed high similarities between culture independent and dependent methods at the phylum level and large differences at the genus level, highlighting the complementarity between the two methods for identification of the native population bacteria in arsenic-rich mine. As a result, the present study can be a resource on microbes for bio-treatment of arsenic in mining waste.

Innovative, ecofriendly biosorbent-biodegrading biofilms for bioremediation of oil- contaminated water.

Immobilization of microorganisms capable of degrading specific contaminants significantly promotes bioremediation processes. In this study, innovative and ecofriendly biosorbent-biodegrading biofilms have been developed in order to remediate oil-contaminated water. This was achieved by immobilizing hydrocarbon-degrading gammaproteobacteria and actinobacteria on biodegradable oil-adsorbing carriers, based on polylactic acid and polycaprolactone electrospun membranes. High capacities for adhesion and proliferation of bacterial cells were observed by scanning electron microscopy. The bioremediation efficiency of the systems, tested on crude oil and quantified by gas chromatography, showed that immobilization increased hydrocarbon biodegradation by up to 23 % compared with free living bacteria. The resulting biosorbent biodegrading biofilms simultaneously adsorbed 100 % of spilled oil and biodegraded more than 66 % over 10 days, with limited environmental dispersion of cells. Biofilm-mediated bioremediation, using eco-friendly supports, is a low-cost, low-impact, versatile tool for bioremediation of aquatic systems.

Modestobacter excelsi sp. nov., a novel actinobacterium isolated from a high altitude Atacama Desert soil.

A polyphasic study was undertaken to establish the taxonomic status of three Modestobacter strains isolated from a high altitude Atacama Desert soil. The isolates, strains 1G6T, 1G14 and 1G50, showed chemotaxonomic and morphological properties characteristic of members of the genus Modestobacter. The peptidoglycan contained meso-diaminopimelic acid, the whole cell sugars were glucose and ribose (diagnostic sugars) and arabinose, the predominant menaquinone was MK-9(H4), polar lipid patterns contained diphosphatidylglycerol, glycophosphatidylinositol, phosphatidylethanolamine (diagnostic component), phosphatidylglycerol and phosphatidylinositol while whole cellular fatty acid profiles consisted of complex mixtures of saturated, unsaturated iso- and anteiso-components. The isolates were shown to have different BOX-PCR fingerprint and physiological profiles. They formed a distinct phyletic line in Modestobacter 16S rRNA gene trees, were most closely related to the type strain of Modestobacter italicus (99.9 % similarity) but were distinguished from this and other closely related Modestobacter type strains using a combination of phenotypic properties. Average nucleotide identity and digital DNA:DNA hybridization similarities between the draft genome sequences of isolate 1G6T and M. italicus BC 501T were 90.9 % and 42.3 %, respectively, indicating that they belong to different species. Based on these phenotypic and genotypic data it is proposed that the isolates be assigned to a novel species in the genus Modestobacter, namely as Modestobacter excelsi with isolate 1G6T (=DSM 107535T =PCM 3004T) as the type strain. Analysis of the whole genome sequence of M. excelsi 1G6T (genome size of 5.26 Mb) showed the presence of genes and gene clusters that encode for properties that are in tune with its adaptation to extreme environmental conditions that prevail in the Atacama Desert biome.

Excision of endosymbiotic bacteria from yeast under aging and starvation stresses.

Although infrequent in our laboratory, growth of bacterial colonies has been observed on top of the purified cultures of yeasts. In this study, the likelihood of bacterial excision from yeast under aging and starvation stresses was assessed using 10 gastric and 10 food-borne yeasts. Yeasts were identified as members of Candida or Saccharomyces genus by amplification and sequencing of D1/D2 region of 26S rDNA. For aging stress, yeasts were cultured on brain heart infusion agar supplemented with sheep blood and incubated at 30 °C for 3-4 weeks. For starvation stress, yeasts were inoculated into distilled water and incubated similarly. After seven days, starved yeasts were cultured on yeast extract glucose agar, incubated similarly and examined daily for appearance of bacterial colonies on top of the yeast's growth. Outgrowth of excised bacteria was observed on top of the cultures of 4 yeasts (Y1, Y3, Y13 and Y18) after 3-7 days. The excised bacteria (B1, B3, B13 and B18) were isolated and identified at the genus level according to their biochemical characteristics as well as amplification and sequencing of 16S rDNA. B1 (Arthrobacter) were excised from Y1 (Candida albicans) upon aging and B3 (Staphylococcus), B13 (Cellulomonas) and B18 (Staphylococcus) were excised from their respective yeasts; Y3 (Candida tropicalis), Y13 (Saccharomyces cerevisiae) and Y18 (Candida glabrata) upon starvation. DNA from yeasts was used for detection of 16S rDNA of their intracellular bacteria and sequencing. Amplified products from yeasts showed sequence similarity to those of excised bacteria. Under normal conditions, yeast exerts tight control on multiplication of its intracellular bacteria. However, upon aging and starvation the control is no longer effective and bacterial outgrowth occurs. Unlimited multiplication of excised bacteria might provide yeast with plenty of food in close vicinity. This could be an evolutionary dialogue between yeast and bacteria that ensures the survival of both partners.

New glycopolymers containing both D- and L-rhamnopyranoses from Rathayibacter iranicus VKM Ac-1602T cell wall.

The cell wall of Rathayibacter iranicus VKM Ac-1602T (family Microbacteriaceae, class Actinobacteria) is characterised by the absence of phosphate-containing and by the presence of two rhamnose-containing glycopolymers. The first is a branched rhamnomannan, in which 60% of mannose residues of the main chain are glycosylated by terminal mannose residues: →2)-α-D-Rhap-(1 → 3)-α-[α-D-Manp-(1 → 6)]-D-Manp-(1 → . The second is a branched teichuronic acid, in which all the rhamnose residues of the main chain are glycosylated by glucose residues:→3)-α-[α-D-Glcp-(1 → 2)]-L-Rhap-(1 → 4)-ß-D-GlcpA-(1 → 2)-α-D-Manp-(1 → 3)-α-D-Galp-(1 → 3)-ß-D-Glcp-(1 → . Both glycopolymers have the unique structures and described in the cell walls of Gram-positive bacteria for the first time. The obtained data allow for a more complete characterisation of the cell wall of the microorganism under investigation and can serve as a phenotypic characterisation of this bacterium. The glycopolymer structures were established using chemical and nuclear magnetic resonance (NMR) spectroscopy methods.

Cell Walls and Membranes of Actinobacteria.

Actinobacteria is a group of diverse bacteria. Most species in this class of bacteria are filamentous aerobes found in soil, including the genus Streptomyces perhaps best known for their fascinating capabilities of producing antibiotics. These bacteria typically have a Gram-positive cell envelope, comprised of a plasma membrane and a thick peptidoglycan layer. However, there is a notable exception of the Corynebacteriales order, which has evolved a unique type of outer membrane likely as a consequence of convergent evolution. In this chapter, we will focus on the unique cell envelope of this order. This cell envelope features the peptidoglycan layer that is covalently modified by an additional layer of arabinogalactan . Furthermore, the arabinogalactan layer provides the platform for the covalent attachment of mycolic acids , some of the longest natural fatty acids that can contain ~100 carbon atoms per molecule. Mycolic acids are thought to be the main component of the outer membrane, which is composed of many additional lipids including trehalose dimycolate, also known as the cord factor. Importantly, a subset of bacteria in the Corynebacteriales order are pathogens of human and domestic animals, including Mycobacterium tuberculosis. The surface coat of these pathogens are the first point of contact with the host immune system, and we now know a number of host receptors specific to molecular patterns exposed on the pathogen's surface, highlighting the importance of understanding how the cell envelope of Actinobacteria is structured and constructed. This chapter describes the main structural and biosynthetic features of major components found in the actinobacterial cell envelopes and highlights the key differences between them.

Reliability of antioxidant potential and in vivo compatibility with extremophilic actinobacterial-mediated magnesium oxide nanoparticle synthesis.

Owing to the hazards of chemical and physical syntheses of magnesium oxide nanoparticles (MgO NPs), an eco-friendly, high-yield, and promising biological method is highly desirable for biomedical applications. Hence, in this study, an extremophilic actinobacterial population (SA8 and SA10) from Salem magnesite mining soil was used as precursors for MgO NP synthesis. The prepared nanoparticles were subjected to X-ray diffraction study and showed face-centred cubic structure with an average particle size of 18-24 nm. Among all, high yield was obtained in SA10 actinobacteria-mediated synthesis of MgO NPs (480 mg/100 mL). In addition, the prepared MgO NPs (10 mg/well) showed 15-17 mm zone of inhibition against bacterial pathogens, especially bigger zones around SA10. The 64.5% antioxidant activity and nonsignificant toxicity of actinobacteria-synthesized MgO NPs in MG-63 cell lines at 100 µg/mL and nonsignificant in vivo toxicity in zebrafish at 0.1 mg/mL were remarkable. In addition, this is the first study to focus on MgO NP synthesis using extremophilic actinobacteria collected from Salem magnesite mining soil for high yield (115 mg/100 mL), reliable with potential antioxidant, and in vitro and in vivo compatibility. These results provided useful information for advanced research and mass production of NP for biomedical applications.

Labeling of Microthrix parvicella in situ: A novel FRET probe based on bisoctyl rhodamine B.

Filamentous bacteria, particularly Microthrix parvicella, are mainly responsible for bulking or foaming of activated sludge. Based on the affinity of M. parvicella to the hydrophobic characteristics of long-chain fatty acids, a novel bisoctyl rhodamine B (BORB) and a novel fluorescence resonance energy transfer (FRET) complex probe were prepared herein to study their properties. When the FRET probe was used in in situ activated sludge, M. parvicella was clearly labeled at 20 nmol/L, which was a reduction of 50 times compared to that of the BORB (1 µmol/L) alone and 500 times compared to the carbazole-quinoline probe reported previously. Compared with fluorescence in situ hybridization, M. parvicella could be clearly labeled using BORB and the FRET probe in situ without requiring complicated pretreatments (i.e., shock and broken process, fixed sample, digestion, and lysozyme treatment). This study discusses the facile approach developed for labeling M. parvicella in early warning expansion, thereby inhibiting and controlling sludge bulking in situ.

Spongiactinospora rosea gen. nov., sp. nov., a new member of the family Streptosporangiaceae.

A novel aerobic, spore-forming, marine actinomycete, designated strain LHW63015T, was isolated from a Craniella marine sponge collected in the South China Sea. The strain formed extensively branched substrate and aerial mycelia which carried long and crooked spore chains composed of ridged spores and spherical pseudosporangia. Strain LHW63015T contained meso-diaminopimelic acid as the diagnostic diamino acid. Glucose, ribose, mannose, galactose and madurose occured in whole-cell hydrolysates. The predominant polar lipids were hydroxyl-phosphatidylethanolamine, phosphoglycolipid and ninhydrin-positive phosphoglycolipid. MK-10(H4) and MK-10(H6) were the predominant menaquinones. The major fatty acids were 10-methyl C17 : 0 and C17 : 1ω8c. The G+C content of the genomic DNA was 70.8 mol%. In phylogenetic analysis based on 16S rRNA gene sequences, strain LHW63015T fell within the family Streptosporangiaceae and formed a distinct monophyletic lineage adjacent to the genus Sphaerisporangium, and shared the highest 16S rRNA gene sequence similarity of 96.2 % with Sphaerisporangium album YIM 48782T. On the basis of the polyphasic evidence, a novel genus and species of the family Streptosporangiaceae, for which the name Spongiactinospora rosea gen. nov., sp. nov., is proposed, with the type strain LHW63015T (=DSM 106635T=CCTCC AA 2018019T).

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

Dynamic bacterial community changes in the autothermal thermophilic aerobic digestion process with cell lysis activities, shaking and temperature increase.

Autothermal thermophilic aerobic digestion (ATAD) is conducted for stabilization of sludge waste and is driven by the action of various microorganisms under aerobic conditions. However, the mechanism controlling bacterial community changes during ATAD via three (initial, middle and final) phases is currently unclear. To investigate this mechanism, activity analysis and a microcosm assay with shaking were performed on a bacterial community during the initial, middle, and final phases of incubation. Cell lysis activities toward gram-negative bacteria, but not gram-positive bacteria, were detected in the ATAD samples in the middle and final phases. During shaking incubation in initial-phase samples at 30 °C, major operational taxonomic units (OTUs) related to Acinetobacter indicus and Arcobacter cibarius dramatically increased along with decreases in several major OTUs. In middle-phase samples at 45 °C, we observed a major alteration of OTUs related to Caldicellulosiruptor bescii and Aciditerrimonas ferrireducens, together with distinct decreases in several other OTUs. Final-phase samples maintained a stable bacterial community with major OTUs showing limited similarities to Heliorestis baculata, Caldicellulosiruptor bescii, and Ornatilinea apprima. In conclusion, the changes in the bacterial community observed during ATAD could be partially attributed to the cell lysis activity toward gram-negative bacteria in the middle and final phases. The microcosm assay suggested that certain physical factors, such as a high oxygen supply and shearing forces, also might contribute to bacterial community changes in the initial and middle phases, and to the stable bacterial community in the final phase of ATAD.

An image analysis-aided method for redundancy reduction in differentiation of identical Actinobacterial strains.

AIM: To simplify the recognition of Actinobacteria, at different stages of the growth phase, from a mixed culture to facilitate the isolation of novel strains of these bacteria for drug discovery purposes. MATERIALS & METHODS: A method was developed based on Gabor transform, and machine learning using k-Nearest Neighbors and Naive Bayes classifier, Logitboost, Bagging and Random Forest to automatically categorize the colonies. RESULTS: A signature pattern was inferred by the model, making the differentiation of identical strains possible. Additionally, higher performance, compared with other classification methods was achieved. CONCLUSION: This automated approach can contribute to the acceleration of the drug discovery process while it simultaneously can diminish the loss of budget due to the redundancy occurred by the inexperienced researchers.

Evaluation of antibacterial potential of mangrove sediment-derived actinomycetes.

Actinomycetes are well-known as the source of bioactive metabolites. In this work, 16 out of 118 (13.6%) isolates of mangrove sediment-derived actinomycetes showed potential antibacterial activity against at least one bacterial strain. Five extracts from isolates AMA11, AMA12 and AMA21 exhibited a broad spectrum antibacterial activity against Staphylococcus aureus ATCC25923, Staphylococcus epidermidis ATCC35984, methicillin-resistant S. aureus (MRSA) SK1, Acinetobacter baumannii NPRC004 and Escherichia coli ATCC25922. Ethyl acetate extract from the cells of AMA11 (AMA11CE) showed high activity against S. aureus and MRSA with the lowest minimum inhibitory concentration (MIC) of 0.5 µg ml-1. At concentration of four times its MIC, AMA11CE destroyed MRSA cells as analysed by the scanning electron microscopy. In addition, AMA11CE, ethyl acetate extract from the culture broth of AMA12 (AMA12BE), AMA12CE and AMA21CE reduced violacein production in Chromobacterium violaceum. Furthermore, at concentrations lower than 10 µg ml-1, all five extracts inhibited biofilm formation by S. epidermidis ATCC35984. The chemical analysis of the most active fraction from AMA11CE by GC-MS revealed the presence of 3-nitro-1,2-benzenedicarboxylic acid, hexadecanoic acid, quinoxaline-2-carboxamide and pentadecanoic acid. The 16S rDNA sequencing analysis revealed that these three potential isolates belonged to the genus Streptomyces. The results revealed that the actinomycetes from mangrove environment would be a good source of bioactive metabolites against pathogenic bacteria.

Characterization and phylogenetic affiliation of Actinobacteria from tropical soils with potential uses for agro-industrial processes.

Secondary metabolites produced by Actinobacteria of tropical soils represent a largely understudied source of novel molecules with relevant application in medicine, pharmaceutical and food industries, agriculture, and environmental bioremediation. The present study aimed to characterize sixty-nine Actinobacteria isolated from compost and tropical soils using morphological, biochemical, and molecular methods. All the isolates showed high variation for morphological traits considering the color of pigments of the aerial and vegetative mycelium and spore chain morphology. The enzymatic activity of amylase, cellulase, and lipase was highly variable. The amylase activity was detected in 53 (76.81%) isolates. Eighteen isolates showed enzymatic index (EI) > 4.0, and the isolates ACJ 45 (Streptomyces curacoi) and ACSL 6 (S. hygroscopicus) showed the highest EI values (6.44 and 6.42, respectively). The cellulase activity varied significantly (P ≤ 0.05) among the isolates. Twenty-nine isolates (42.02%) showed high cellulase activity, and the isolates ACJ 48 (S. chiangmaiensis) and ACJ 53 (S. cyslabdanicus) showed the highest EI values (6.56 for both isolates). The lipase activity varied statistically (P ≤ 0.05) with fourteen isolates (20.29%) considered good lipase producers (EI > 2.0). The isolate ACSL 6 (S. hygroscopicus) showed the highest EI value of 2.60. Molecular analysis of partial 16S rRNA gene sequencing revealed the existence of 49 species, being 38 species with only one representative member and 11 species represented by one or more strains. All species belonged to three genera, namely Streptomyces (82.61%), Amycolatopsis (7.25%), and Kitasatospora (10.14%). The present results showed the high biotechnological potential of different Actinobacteria from tropical soils.

Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria.

UNLABELLED: Dividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond) daughter cell separation (DCS) driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes) or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives), observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis) with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelae IMPORTANCE: Much of our knowledge on bacterial cytokinesis comes from studying rod-shaped model organisms such as Escherichia coli and Bacillus subtilis Less is known about variations in this process among different bacterial species. While cell division in many bacteria has been characterized to some extent genetically or biochemically, few species have been examined using video microscopy to uncover the kinetics of cytokinesis and daughter cell separation (DCS). In this work, we found that fast (millisecond) DCS is exhibited by species in two independent clades of Gram-positive bacteria and is particularly prevalent among the Actinobacteria, a diverse group that includes significant pathogens as well as bacteria that generate medically important antibiotics.

Building the bacterial cell wall at the pole.

Polar growth is the predominant mode of cell wall extension in the Actinobacteria and the alphaproteobacterial clade Rhizobiales. The observation of polar elongation in taxonomically diverse bacteria suggests that polar growth may have evolved independently. Indeed, the regulatory mechanisms governing the assembly of cell wall biosynthesis machinery at the pole are distinct in the Actinobacteria and Rhizobiales. Here we highlight recent advances in our understanding of polar growth mechanisms in bacteria, with an emphasis on Streptomyces and Agrobacterium. This review illustrates that common themes are emerging in the regulation of polar growth in diverse bacteria. Emerging themes include the use of landmark proteins to direct growth to the pole and coordination of polar growth with cell-cycle progression.

In situ microscopy as a tool for the monitoring of filamentous bacteria: a case study in an industrial activated sludge system dominated by M. parvicella.

The present study demonstrates the application of in situ microscopy for monitoring the growth of filamentous bacteria which can induce disturbances in an industrial activated sludge process. An in situ microscope (ISM) is immersed directly into samples of activated sludge with Microthrix parvicella as dominating species. Without needing further preparatory steps, the automatic evaluation of the ISM-images generates two signals: the number of individual filaments per image (ISM-filament counting) and the total extended filament length (TEFL) per image (ISM-online TEFL). In this first version of the image-processing algorithm, closely spaced crossing filament-segments or filaments within bulk material are not detected. The signals show highly linear correlation both with the standard filament index and the TEFL. Correlations were further substantiated by comparison with real-time polymerase chain reaction (real-time PCR) measurements of M. parvicella and of the diluted sludge volume index. In this case study, in situ microscopy proved to be a suitable tool for straightforward online-monitoring of filamentous bacteria in activated sludge systems. With future adaptation of the system to different filament morphologies, including cross-linking filaments, bundles, and attached growth, the system will be applicable to other wastewater treatment plants.