Lack Of Cell Wall Research Articles

Mycoplasma bovis mastitis in dairy cattle.

Mycoplasma bovis has recently been identified increasingly in dairy cows causing huge economic losses to the dairy industry. M. bovis is a causative agent for mastitis, pneumonia, endometritis, endocarditis, arthritis, otitis media, and many other clinical symptoms in cattle. However, some infected cows are asymptomatic or may not shed the pathogen for weeks to years. This characteristic of M. bovis, along with the lack of adequate testing and identification methods in many parts of the world until recently, has allowed the M. bovis to be largely undetected despite its increased prevalence in dairy farms. Due to growing levels of antimicrobial resistance among wild-type M. bovis isolates and lack of cell walls in mycoplasmas that enable them to be intrinsically resistant to beta-lactam antibiotics that are widely used in dairy farms, there is no effective treatment for M. bovis mastitis. Similarly, there is no commercially available effective vaccine for M. bovis mastitis. The major constraint to developing effective intervention tools is limited knowledge of the virulence factors and mechanisms of the pathogenesis of M. bovis mastitis. There is lack of quick and reliable diagnostic methods with high specificity and sensitivity for M. bovis. This review is a summary of the current state of knowledge of the virulence factors, pathogenesis, clinical manifestations, diagnosis, and control of M. bovis mastitis in dairy cows.

Five questions on the cell-to-cell movement of Orthotospoviruses

Plant viruses employ Movement proteins (MP) for their cell to cell spread through plasmodesmata (PD). MP modifies the PD and increases its size exclusion limit (SEL). However, the mechanism by which MPs are targeted to the PD is still unresolved and there is a lack of consensus owing to limited studies on their biochemical and structural characters. The non structural protein m (NSm) functions as the MP in Orthotospoviruses. Tospoviral NSm associate with ER membrane. They also form tubules in protoplasts. Groundnut bud necrosis virus (GBNV), a tospovirus, infects several crop plants throughout India and is economically very important. GBNV NSm associates with the membrane strongly via the C-terminal coiled-coil domain, modifies the membrane and causes vesicle fusion in vitro and remodels the ER network into vesicles in vivo. These vesicles are in contrast to the tubules formed by other related tospovirus in cells lacking cell wall. In this review, five important questions on the cell-to-cell movement of tospoviruses have been addressed and based on the various reports, a plausible model on the cell-to-cell movement of Orthotospoviruses is presented.

Mycoplasma bovis and Mannheimia haemolytica dynamics during acute bovine respiratory disease in feedlot cattle

Mycoplasma bovis is a bacterium member of the family Myco­plasmataceae, characterized by a small genome, lack of cell wall, and high nutritional requirements for in vitro growth (Li et al., 2011; Parker et al., 2018; Dudek et al., 2020). Traditionally, M. bovis has been linked to chronic BRD cases and treatment failures (Booker et al., 2008; Hermeyer et al., 2012; Gershwin et al., 2015; Becker et al., 2020). Several virulence factors have been implicated in M. bovis ability to persist in the lungs of cat­tle with BRD, such as variable surface proteins (Vsp), adhesins, nucleases, H2O2 production and biofilm formation (Burki et al., 2015; Perez-Casal, 2020). Due to the difficulties of growing M. bovis in the lab, the inherent culture bias (Prakash et al., 2021) could have shaped our current knowledge of M. bovis in BRD.
 Recent evidence has emerged linking M. bovis presence in the upper respiratory tract with acute BRD status (Timsit et al., 2018; Centeno-Martinez et al., 2022). However, the association between M. bovis and other BRD agents, especially Mannheim­ia haemolytica, over time remains unknown during the first month on the feedlot. A more comprehensive understanding of the dependencies among BRD pathogens over time will help develop new non-antibiotic control strategies and subsequently reduce the burden of BRD on beef cattle production. Therefore, this study aimed to evaluate the association of M. bovis and M. haemolytica during acute BRD in feedlot cattle.

Rejection and fouling of track-etched microfiltration membranes by Acholeplasma laidlawii: Clues to mycoplasma behavior during “sterile” dead-end filtration

Mycoplasma contamination is commonplace during processing of biopharmaceuticals necessitating its sterile filtration. Consequently, we measured the passage of Acholeplasma laidlawii (a small bacterium lacking cell wall like mycoplasma) across track-etched membranes rated at 100 nm and 220 nm that are designated as “sterile filters” and analyzed the entire progression of fouling beginning with pore occlusion before transitioning to a cake. Initial biofouling was quantified to be intermediate blocking before bacteria formed highly compressible cakes. Under identical experimental conditions, phase inversion membranes of the same nominal pore rating rejected A. laidlawii to a significantly greater extent suggesting depth filtration and capture within the polymer matrix in contrast to surface capture by their track-etched counterparts. A facile fluorescence microscopy method to rapidly detect slow-growing A. laidlawii cells was demonstrated under conditions of low retention. Evidence of cake relaxation by depressurizing the filter and subsequent mobilization of individual cells upon re-pressurization is given. Multiple lines of evidence demonstrated the pressure-induced deformation of individual cells resulting in their incomplete rejection by membranes with pore ratings smaller than the bacterium's unstressed diameter: (i) complete retention of rigid silica colloids of similar size, shape, and zeta potential regardless of pressure in contrast to incomplete A. laidlawii removals, (ii) reduced bacterial removal by both membranes with increasing filtration pressure, (iii) 150–600 fold less permeable bacterial cakes at any given pressure compared with silica cakes, and (iv) presence of flattened and irregularly shaped A. laidlawii cells in electron micrographs whereas they were near perfect cocci at atmospheric pressure.

Pulsed light treatment of table grape juice: Influence of matrix pH on microbial and enzyme inactivation kinetics

The influence of pulsed light (PL) treatment and matrix pH was tested in the case of green table grape juice. Escherichia coli, Listeria monocytogenes, and Saccharomyces cerevisiae were inoculated into the table grape juices with matrix pH levels of 3.0, 3.5, and 4.0. Further, the juice was treated at 378–3186 J/cm2. The Weibull model better explained the microbial inactivation data. The shape parameters (β) from the Weibull model for E. coli, L. monocytogenes, and S. cerevisiae are 0.95 ± 0.02, 0.55 ± 0.04, and 0.40 ± 0.03, respectively. A lesser δ-value (scale parameter in J/cm2) was obtained for each microorganism at a lower pH. PL exposure resulted in a lack of cell visibility, cell wall, and cell membrane disruption in bacterial cells. The inactivation of polyphenol oxidase (PPO), peroxidase (POD), and pectin methyl esterase (PME) was studied at an elevated fluence level of 1152–3186 J/cm2. Enzymes in the juice were less sensitive to PL than microorganisms. The enzyme inactivation of >90% was achieved at 3186 J/cm2; thus, the juice was adjudged as the microbially safe and enzymatically stable pasteurized sample. The PL-induced enzyme inactivation followed nth-order kinetics with an inactivation order (n) of 1.1, 1.15, and 0.85 for PPO, POD, and PME, respectively. The enzyme inactivation rate was higher at a low pH. The PL-treated pasteurized juice at pH 3.5 showed the highest sensory acceptance (7.4 out of 9), with a minimal color change, a 7% loss in total phenolics, and a 12% loss in vitamin C and flavonoids.

Immune Response and Histological Changes in Broilers Chickens Vaccinated with Mycoplasma gallisepticum Vaccines.

Mycoplasma is unique among prokaryotes because of its small size, small genomes, and complete lack of cell walls, which makes them cell wall-less prokaryotes. This study aimed to evaluate the effect of vaccinating one-day-old chicks with inactivated and live vaccines (CRDF) of Mycoplasma gallisepticum (MG) on their humoral immune response and immune organs. The Enzyme-Linked Immunosorbent Assay was used to measure Ab titers and investigate histopathological changes. A total of 130 one-day-old broiler chicks were randomly divided into four groups of 30. The groups were treated as follows: G1 included the chicks vaccinated with live F-strain MG vaccine (on eye drop of 0.03ml/dose), G2 included the chicks vaccinated with inactivated MG (0.3 ml s.c) vaccine, G3 included the chicks vaccinated with inactivated and live MG vaccines, and G4 was considered the control group, in which the chicks were not vaccinated. Blood samples were collected on days 21 and 35 of the chick's life to measure the titers of specific antibodies. On day 35, the chicks were dissected, and the bursa of Fabricius, as well as the spleen, were removed for histological evaluations. On day 21, the results showed a significant difference (P≤0.05) between all vaccinated groups in Ab titers, compared to G4, with the highest mean in G3, followed by G2 and G1, in descending order. On day 35, there was a significant difference (P≤0.05) between G3 and other vaccinated groups (G2 and G1), as well as G4. In addition, there was a significant increase in all vaccinated groups on day 35, compared to day 21. In G1, histopathological examination results showed a moderate lymphocytic hyperplasia bursal follicle. In G2, varying degrees of lymphoproliferative were observed in the major bursal follicle, and in G3, a marked lymphocytic hyperplasia bursal follicle was observed. In G4, on the other hand, no obvious histopathological findings were recorded. The results of the spleen histopathological evaluation showed various degrees of lymphoproliferative and moderate neutrophilic infiltrate in the red pulp in G1, and mild sinus congestion with scattered lymphocytes was recorded in the lumen in G2. In the spleen of the chicks in G3, reactive lymphoid hyperplasia was observed. In contrast to the groups mentioned above, in G4, the spleen structure showed a typical structure. It was concluded that the chicks vaccinated with inactivated and live MG vaccines experienced increased production of Ab titers and the immune stimulation of immune organs.

Applications of comet and MTT assays in studying Dunaliella algae species

Chloroplasts and unicellular algae genomes, are the most important and vital intracellular organelles which determine the cells physiological performance. Using Comet and MTT assay technics in algal studies, will provide researchers prominent features about both their photosynthesis and genomes. Using two unicellular algal groups, Dunaliella salina (orange) and Dunaliella viridis (green), collected from a hypersaline lake (Maharloo lake) located east of Shiraz, Iran, MTT and Comet assays along with their trouble shootings were used to study these two algae. Results showed that, MTT was reduced due to the presence of oxidoreductive enzymes both in photosynthetic reaction centers and in electron transport chains. It was concluded that, MTT reduction in unicellular algal cells, mostly reflects the chloroplasts activity. The rates of light absorbance at λ = 570 nm due to MTT reduction for 100 000 D. salina cells/well (R2 = 0.9457) as well as for 160 000 D. viridis cells/well (R2=9838) were the same as equal to 1. Due to the presence of beta-carotene in D. salina cells, lower number of cell was required to give the same absorbance as compared to D. viridis. In continuation of this study, non-physiological apoptosis cells death caused by Cis‑platinum drug was investigated in these two algal species. Using Comet assay, the DNA damage was determined in both positive and negative control groups. The tail averages in positive control group and in negative control group, were 14.9 % and 0.73 % respectively. Unicellular aquatic algae that lack cell wall, are suitable models to be used in such studies. We would like to recommend the application of MTT and Comet assays technics due to their high sensitivities for revealing damages done to both photosynthetic system and to DNA which will be effective tools for monitoring the biological and non-biological environmental hazards.

First report of a ‘Candidatus Phytoplasma australasia’‐related strain (16SrII‐D) associated with crown choking of areca palm in India

Areca palm (Areca catechu) is an economically important crop that is grown primarily in South Asia and South India, mainly in the states of Karnataka, Kerala and Maharashtra. With 0.73 million hectares cultivated and 1.2 million tonnes produced, India ranks first in world areca nut production. The state of Karnataka alone accounts 80% of annual production in India. During January 2021, crown choking symptoms (locally named “Hidimundige”), which resemble phytoplasma symptoms were observed on areca palm plantations. The highest disease incidence (50%) was recorded in Ajjarani, Sirsi Taluk and Uttara Kannda, followed by 30% in Haralakatta, Channagiri Taluk, Davanagere, Kelaginuru and Honnavara Taluk, in the district of Uttara Kannada (Karnataka state). The lowest disease incidence was 1% for the Dakashina Kannada and Tumkur districts in Karnataka.The most prominent symptom in infected palms was short, dark green leaves with a wavy leaf lamina. Leaflets failed to unfold properly leading to a bunchy top or witches’ broom appearance, and became crinkled and brittle. Reduced internodal length, drying of inflorescences and tapering of stem at the apex were also recorded. The roots became black, crinkled and brittle, and a pinkish or brownish discoloration was observed in the phloem. Affected plants failed to produce new leaves and existing leaves fell leading to a bare stem and death of the plant in quick succession (Figs. 1-3). About 80–100% of the plants died in affected areas. Seven leaf samples were collected from diseased palms growing in the Zonal Agricultural & Horticultural Research Station, Keladi Shivappa Nayaka University of Agricultural and Horticultural Sciences in Shivamogga, Karnataka (Latitude: 13° 55' 47.748“. Longitude: 75° 34' 5.1636”) during March 2022. Typical phytoplasma pleomorphic bodies lacking cell walls were seen in the sieve tube cells of leaf midrib sections from diseased areca palms, when examined by transmission electronic microscopy (Spurr, 1969) at the Sri Sai Histology Center, Hyderbad India (Fig. 4). The phytoplasma cell sizes ranged from 500 - 2000 nm. Seven leaf and three inflorescence samples from diseased palms and two leaf samples from asymptomatic palms were collected from the same location. Total DNA was extracted by the CTAB method and subjected to direct PCR using primers P1 (Deng & Hiruki, 1991) and P7 (Schneider et al., 1995) and nested PCR with R16F2n/R16R2 primers (Gundersen & Lee, 1996) that amplify the phytoplasma 16S rRNA gene. The expected direct (1.8 Kb) and nested (1.25 Kb) PCR products were obtained from all the diseased plant samples and positive reference controls but not from the symptomless plants. The R16F2n/R2 PCR products were cloned and sequenced directly at Biokart Pvt. Ltd., Karnataka, India, and the sequence deposited in GenBank (Accession No. ON606488). The R16F2n/R2 sequence was subjected to BLAST analysis and showed the highest nucleotide identity (99.92%) with 16SrII phytoplasma isolates, ‘Candidatus Phytoplasma aurantifolia’ (MT555412, MT555411) identified in Croton bonplandianus in Maharashtra, India. Virtual RFLP analysis of the R16F2n/R2 sequence with iPhyClassifier yielded an RFLP pattern identical to that of the 16Sr group II, subgroup D (Y10097) with a similarity coefficient of 1.00. Phylogenetic analysis (MEGA 7.0.18) using the neighbour-joining algorithm with selected phytoplasma reference strains confirmed that the areca palm phytoplasma clustered within the clade of phytoplasma strains of the 16SrII group (Fig. 5). In India, the cultivation of areca nut is mostly limited to the states of Assam, Karnataka and Kerala, and palms in these areas are affected by a yellowing disease named yellow leaf disease (YLD). YLD is one of the most important diseases of this palm species (Nambiar, 1949) and it is associated with the 16SrXI phytoplasma group, ‘Ca. P. oryzae’. (Chaithra et al., 2014). ‘Ca. P. aurantifolia’ is known to infect several plant species in India (Rao et al., 2017). However, this is the first report of a ‘Ca. P. aurantifolia’- related strain causing crown choking of Areca catechu in India. The impact of crown choking on areca nut yield, the geographic distribution of the disease and its epidemiological implication in India are currently under investigation.

Genetic Manipulation of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a small cell wall-lacking bacterium that is a common cause of bronchitis and pneumonia in humans. In addition to its clinical importance, M. pneumoniae has recently been considered a promising model organism for synthetic biology because of its small genome size and unique cell structure. At one cell pole, M. pneumoniae forms the attachment organelle that is responsible for adherence to host cells and gliding motility. The attachment organelle is a membrane protrusion and is composed of number of molecules, including adhesin and cytoskeletal proteins. Genetic manipulation techniques are key research approaches for understanding the structure and the function of this unique molecular machinery. In this chapter, standard genetic engineering methods for this species using the Tn4001 transposon vector are described.

Lack of evidence for Mycoplasma spp. in bulk tank milk of herds located in mid-western Colombia

Mycoplasma spp. is reported as a highly contagious mastitis-causing bacteria in dairy cattle, without successful or low response to most common antibiotic treatments due to the lack of cell wall. In Colombia it has been reported in the Central Andean region during 2014. The aim was to estimate the prevalence of Mycoplasma spp. in bulk tank milk using microbiological and molecular diagnosis. A random longitudinal study en­rolling 220 commercial dairy farms located in four provinces of the mid-western region of Colombia from four pasteurizer companies was performed. Bulk tank milk samples were collected once monthly for three months period for determining somatic cell count (SCC) and microbiological and molecular diagnosis of Mycoplasma spp. cultures were done without pre-enrichment procedures directly in mycoplasma agar with cefoperazone to inhibit growth of opportunistic microorganisms, plates were incubated under 37° C and atmosphere of 10% CO2 and inspected during a 10d period. Molecular analysis was done by a multiplex PCR using specific primers targeting the 16S-23S rARN gene of Mycoplasma spp. and from non-pathogenic bacteria occasionally found in milk. LnSCC average of included dairy farms was 6.19 x103 cells/mL, Mycoplasma spp. was not isolated during microbiological cultures, and no DNA belonging to the species was detected by PCR in the 220 bulk tanks milk, with an estimated prevalence lower than 2.3%. This finding shows that there is not microbiological or molecular evidence that demonstrates the presence of the pathogen in the milk from the mid-western region of Colombia at herd level.

Detection and characterization of rice orange leaf phytoplasma infection in rice and Recilia dorsalis

Phytoplasmas are small bacterial parasites that lack cell walls and are transmitted in a persistent-propagative manner by insect vectors. However, detailed multiplication patterns and movements of phytoplasmas within host plant and insect vector remain elusive. In this study, a specific antibody against the immunodominant membrane protein (Imp) of rice orange leaf phytoplasma (ROLP) was generated and subjected to the frozen section immune gold labeling assay, immune gold labeling microscopy, and immune fluorescence labeling assay to investigate how ROLP enters, propagates, and spreads in rice and its leafhopper vector Recilia dorsalis at the ultrastructural level. During acquisition and transmission by insect vectors, ROLPs could squeeze and penetrate the multiple membrane/tissue barriers such as microvilli, apical plasmalemma, and basal lamina in the intestines or salivary glands by endocytosis- and exocytosis-like mechanism. Furthermore, ROLP infection is restricted to the type IV and V cells of salivary glands. In contrast to the classical binary fission used by most bacteria, electron microscopy reveals that ROLP exploits an asymmetrical budding strategy to replicate in plant hosts and insect vectors. In this process, a cellular protrusion of ROLP forms a bud to reproduce the offspring by fission at the junction between the bud and the ROLP main body. These results clarify the infection characteristics of ROLP in rice and R. dorsalis, which will help guide the formulation of ROLP prevention and control strategies.

A Rapid and Efficient Method for Isolation and Transformation of Cotton Callus Protoplast.

Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12–16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts.

Amphibian mucus triggers a developmental transition in the frog-killing chytrid fungus

The frog-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is decimating amphibian populations around the world.1-4Bd has a biphasic life cycle, alternating between motile zoospores that disperse within aquatic environments and sessile sporangia that grow within the mucus-coated skin of amphibians.5,6 Zoospores lack cell walls and swim rapidly through aquatic environments using a posterior flagellum and crawl across solid surfaces using actin structures similar to those of human cells.7,8Bd transitions from this motile dispersal form to its reproductive form by absorbing its flagellum, rearranging its actin cytoskeleton, and rapidly building a chitin-based cell wall-a process called "encystation."5-7 The resulting sporangium increases in volume by two or three orders of magnitude while undergoing rounds of mitosis without cytokinesis to form a large ceonocyte. The sporangium then cellurizes by dividing its cytoplasm into dozens of new zoospores. After exiting the sporangium through a discharge tube onto the amphibian skin, daughter zoospores can then reinfect the same individual or find a new host.5 Although encystation is critical to Bd growth, whether and how this developmental transition is triggered by external signals was previously unknown. We discovered that exposure to amphibian mucus triggers rapid and reproducible encystation within minutes. This response can be recapitulated with purified mucin, the bulk component of mucus, but not by similarly viscous methylcellulose or simple sugars. Mucin-induced encystation does not require gene expression but does require surface adhesion, calcium signaling, and modulation of the actin cytoskeleton. Mucus-induced encystation may represent a key mechanism for synchronizing Bd development with the arrival at the host.

A natural mutation of the NST1 gene arrests secondary cell wall biosynthesis in the seed coat of a hull-less pumpkin accession.

Hull-less pumpkins (Cucurbita pepo L.) are naturally occurring novel variants known as oilseed or naked-seeded pumpkins, and are characterized by the absence of a normal lignified seed coat. Due to a specialized seed coat structure, these variants serve as a good model for studying seed coat formation and simplify the processing of pumpkin seeds. However, causal genes for this hull-less trait still remain unknown. Here, by bulked segregant analysis and fine mapping, we found that mutation of a single gene, NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 (NST1), accounts for the hull-less trait. A 14-bp sequence insertion in the CpNST1 gene causes premature termination of CpNST1 translation, leading to lack of secondary cell wall (SCW) biosynthesis in hull-less seed coats. In situ hybridization analysis provided further evidence for the role of CpNST1 in pumpkin seed coat SCW biosynthesis. Interestingly, through secondary cell wall compositional analysis, we found that the main SCW components differed among cell layers in the seed coat. RNA-seq analysis indicated an upstream role of CpNST1 in the SCW biosynthesis network. Collectively, our findings provide mechanistic insight into seed coat SCW biosynthesis, and a target gene for breeders to introduce this hull-less trait for commercial exploitation.

Mycoplasma bovis Mastitis.

Bovine mycoplasmoses, which is mostly caused by Mycoplasma bovis, is a significant problem in the dairy and beef industry. Mycoplasmal mastitis has a global occurrence with notable effects in the United States and Europe. The pathogen was first detected in a mastitis case in California, United States, and regarded as major contagious mastitis. It is highly contagious and resistant to antibiotics and lack cell wall rendering certain group of antibiotics ineffective. Outbreaks mostly originate from introduction of diseased dairy cows to a farm and poor hygienic practices that help to maintain cow to cow transmission. Rapid detection scheme is needed to be in place in dairy farms to devise preventive measures and stop future outbreaks. However; early detection is hampered by the fastidious growth of M. bovis and the need for specialized equipment and reagents in laboratory settings. Intramammary Mycoplasma bovis infections cause elevation in milk somatic cell count which is one of the important factors to determine milk quality for grading and hence dictates milk price. There are multiple attributes of M. bovis regarded as virulence factors such as adhesion to and invasion into host cells, avoidance of phagocytosis, resistance to killing by the alternative complement system, biofilm formation, and hydrogen peroxide production. Nevertheless, there are still undetermined virulence factors that hamper the development of sustainable control tools such as effective vaccine. To date, most vaccine trials have failed, and there is no commercial M. bovis mastitis vaccine. Mycoplasma bovis has been shown to modulate both humoral and cellular immune response during bovine mastitis. In the future, research seeking new immunogenic and protective vaccine targets are highly recommended to control this important dairy cattle disease worldwide.

The wall-less bacterium Spiroplasma poulsonii builds a polymeric cytoskeleton composed of interacting MreB isoforms

SummaryA rigid cell wall defines the morphology of most bacteria. MreB, a bacterial homologue of actin, plays a major role in coordinating cell wall biogenesis and defining cell shape. Spiroplasma are wall-less bacteria that robustly grow with a characteristic helical shape. Paradoxal to their lack of cell wall, the Spiroplasma genome contains five homologs of MreB (SpMreBs). Here, we investigate the function of SpMreBs in forming a polymeric cytoskeleton. We found that, in vivo, Spiroplasma maintain a high concentration of all MreB isoforms. By leveraging a heterologous expression system that bypasses the poor genetic tractability of Spiroplasma, we found that SpMreBs produced polymeric filaments of various morphologies. We characterized an interaction network between isoforms that regulate filament formation and patterning. Therefore, our results support the hypothesis where combined SpMreB isoforms would form an inner polymeric cytoskeleton in vivo that shapes the cell in a wall-independent manner.

Non culturable bacteria associated with COVID-19: More details are demanded

Coronavirus disease 2019 (COVID-19) is a zoonotic infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the extreme acute respiratory syndrome. Coronavirus is a zoonotic infection with a positive polarity RNA envelope that belongs to the Coronaviridae family. There are four recognized genera of coronavirus, however on January 10, 2020, a new coronavirus arose in Wuhan, China, causing a serious pulmonary outbreak. Severe acute respiratory syndrome coronavirus 2 appears to be the third highly deadly human coronavirus to develop in the recent two decades, following SARS coronavirus and Middle East respiratory syndrome (MERS) coronavirus. Severe acute respiratory syndrome coronavirus 2 infection associated with a bacterial pathogen (combined viral and bacterial pneumonia) was described in COVID-19. Secondary bacterial pneumonia can develop after a viral respiratory illness has cleared up or during the healing period. In fatal instances, bacterial infections are more prevalent than in recovered patients. If bacterial infections are the cause of death in COVID-19, this has significant implications for patient treatment. The death rate for patients who acquired secondary infections was 56.7 %, compared to 10.6 % for all COVID-19 patients hospitalized. In 78 % of the cases, Gram-negative bacteria were found. The most common pathogen was Klebsiella pneumoniae (29 %), followed by Acinetobacter baumannii (21%). Pseudomonas aeruginosa (9.6%) and Escherichia coli (8.2%). methicillin-resistant Staphylococcus aureus was isolated only in 1.29%. High levels of carbapenem resistance were seen in Acinetobacter baumannii (92.6%) followed by Klebsiella pneumoniae (72.8%). According to recent research, bacterial co-infection occurred in 3.1–3.5% of COVID-19 patients at admission, with subsequent bacterial infections occurring in up to 15% of patients following hospitalization. Atypical organisms linked to COVID-19 are organisms that are difficult to grow and do not show up on a gram stain. Because antibiotics must be able to enter intracellularly to reach their intended target, they are difficult to separate and treat due to their intracellular nature. Because atypical organisms lack cell walls, such as Mycoplasma pneumoniae, the most prevalent, beta-lactam antibiotics are not indicated. Antibiotic resistance, poor compliance, and an inability to take oral medicines are all factors that contribute to treatment failure. Furthermore, some individuals may have obstructive lung lesions or a misdiagnosis. Atypical pneumonia can be contracted from a variety of causes. Mycoplasma pneumoniae, which is associated with close living conditions such as at school and military barracks, legionella from stagnant water sources, Chlamydophila pneumoniae, Coxiella brunette, and Francisella tularensis from various mammalian sources are among the most commonly identified atypical pathogens. Conclusion: Secondary infections in COVID-19 patients have been linked to poor health outcomes. Non-culturable bacteria have been associated with death in COVID-19 patients, and they may require a specific antibiotic to preserve the patient's life. More research is needed to validate this issue, as there is currently little evidence available.

A possible billion-year-old holozoan with differentiated multicellularity

Sediments of the Torridonian sequence of the Northwest Scottish Highlands contain a wide array of microfossils, documenting life in a non-marine setting a billion years ago (1 Ga).1-4 Phosphate nodules from the Diabaig Formation at Loch Torridon preserve microorganisms with cellular-level fidelity,5,6 allowing for partial reconstruction of the developmental stages of a new organism, Bicellum brasieri gen. et sp. nov. The mature form of Bicellum consists of a solid, spherical ball of tightly packed cells (a stereoblast) of isodiametric cells enclosed in a monolayer of elongated, sausage-shaped cells. However, two populations of naked stereoblasts show mixed cell shapes, which we infer to indicate incipient development of elongated cells that were migrating to the periphery of the cell mass. These simple morphogenetic movements could be explained by differential cell-cell adhesion.7,8 In fact, the basic morphology of Bicellum is topologically similar to that of experimentally produced cell masses that were shown to spontaneously segregate into two distinct domains based on differential cadherin-based cell adhesion.9 The lack of rigid cell walls in the stereoblast renders an algal affinity for Bicellum unlikely: its overall morphology is more consistent with a holozoan origin. Unicellular holozoans are known today to form multicellular stages within complex life cycles,10-13 so the occurrence of such simple levels of transient multicellularity seen here is consistent with a holozoan affinity. Regardless of precise phylogenetic placement, these fossils demonstrate simple cell differentiation and morphogenic processes that are similar to those seen in some metazoans today.

Cell-Wall-Deficient Bacteria in Oral Biofilm: Association with Periodontitis

Cell-wall-deficient bacteria are those that lack cell walls and live in a pleomorphic state. The genus Mycoplasma and L-form bacteria are both members of this group. The aim of this study was to search cellwall-deficient bacteria in periodontal biofilm and link their presence to periodontal disease. Eighty-nine individuals were recruited and divided into three groups: periodontally healthy individuals, individuals with chronic periodontitis, and those with aggressive periodontitis. The presence of cell-wall-deficient bacteria was detected in freshly collected biofilm by light microscopy, transmission electron microscopy (TEM) with and without electron microscopy in situ hybridization, atomic force microscopy and DNA stain (Hoechst). A new dichotomic index of classification for prevalence and morphologic variants was developed to classify cell-wall-deficient bacteria in periodontal biofilm. Cell-wall-deficient bacteria were found in periodontal biofilm and classified into protoplastic, everted, filament and intracellular forms, the last one mainly associated with aggressive periodontitis. We also assessed the prevalence of periodontopathic bacteria by means of polymerase chain-reaction (PCR) and found no clear, statistically significant, correlation among periodontal pathogens tested (except T. denticola) that allowed individuals with chronic periodontitis to be distinguished from those with aggressive periodontitis. Association between cell-walldeficient bacteria and periodontal condition was: periodontally healthy, 3.3% (1/30); individuals with chronic periodontitis, 30.6% (11/36); and those with aggressive periodontitis, 100% (23/23). Cell-walldeficient bacteria were detected in periodontal biofilm and linked to aggressive periodontitis.

Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts.

Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.