Planctoellipticum variicoloris gen. nov., sp. nov., a novel member of the family Planctomycetaceae isolated from wastewater of the aeration lagoon of a sugar processing plant in Northern Germany.

In the present study, we characterise a strain isolated from the wastewater aeration lagoon of a sugar processing plant in Schleswig (Northern Germany) by Heinz Schlesner. As a pioneer in planctomycetal research, he isolated numerous strains belonging to the phylum Planctomycetota from aquatic habitats around the world. Phylogenetic analyses show that strain SH412T belongs to the family Planctomycetaceae and shares with 91.6% the highest 16S rRNA gene sequence similarity with Planctopirus limnophila DSM 3776T. Its genome has a length of 7.3 Mb and a G + C content of 63.6%. Optimal growth of strain SH412T occurs at pH 7.0-7.5 and 28 °C with its pigmentation depending on sunlight exposure. Strain SH412T reproduces by polar asymmetric division ("budding") and forms ovoid cells. The cell size determination was performed using a semi-automatic pipeline, which we first evaluated with the model species P. limnophila and then applied to strain SH412T. Furthermore, the data acquired during time-lapse analyses suggests a lifestyle switch from flagellated daughter cells to non-flagellated mother cells in the subsequent cycle. Based on our data, we suggest that strain SH412T represents a novel species within a novel genus, for which we propose the name Planctoellipticum variicoloris gen. nov., sp. nov., with strain SH412T (= CECT 30430T = STH00996T, the STH number refers to the Jena Microbial Resource Collection JMRC) as the type strain of the new species.

Elongation at Midcell in Preparation of Cell Division Requires FtsZ, but Not MreB nor PBP2 in Caulobacter crescentus.

Controlled growth of the cell wall is a key prerequisite for bacterial cell division. The existing view of the canonical rod-shaped bacterial cell dictates that newborn cells first elongate throughout their side walls using the elongasome protein complex, and subsequently use the divisome to coordinate constriction of the dividing daughter cells. Interestingly, another growth phase has been observed in between elongasome-mediated elongation and constriction, during which the cell elongates from the midcell outward. This growth phase, that has been observed in Escherichia coli and Caulobacter crescentus, remains severely understudied and its mechanisms remain elusive. One pressing open question is which role the elongasome key-component MreB plays in this respect. This study quantitatively investigates this growth phase in C. crescentus and focuses on the role of both divisome and elongasome components. This growth phase is found to initiate well after MreB localizes at midcell, although it does not require its presence at this subcellular location nor the action of key elongasome components. Instead, the divisome component FtsZ seems to be required for elongation at midcell. This study thus shines more light on this growth phase in an important model organism and paves the road to more in-depth studies.

An unexpected puzzle piece links polarity and chromosome segregation.

To ensure successful daughter cell production with one chromosome each, C. crescentus bacteria use an extensively regulated phosphorelay to link all involved cellular processes. In this issue of Developmental Cell, Guzzo et al. (2021) show that the activity of this phosphorelay depends on the translocation of the segregating chromosome.

Learning the distribution of single-cell chromosome conformations in bacteria reveals emergent order across genomic scales.

The order and variability of bacterial chromosome organization, contained within the distribution of chromosome conformations, are unclear. Here, we develop a fully data-driven maximum entropy approach to extract single-cell 3D chromosome conformations from Hi-C experiments on the model organism Caulobacter crescentus. The predictive power of our model is validated by independent experiments. We find that on large genomic scales, organizational features are predominantly present along the long cell axis: chromosomal loci exhibit striking long-ranged two-point axial correlations, indicating emergent order. This organization is associated with large genomic clusters we term Super Domains (SuDs), whose existence we support with super-resolution microscopy. On smaller genomic scales, our model reveals chromosome extensions that correlate with transcriptional and loop extrusion activity. Finally, we quantify the information contained in chromosome organization that may guide cellular processes. Our approach can be extended to other species, providing a general strategy to resolve variability in single-cell chromosomal organization.

Generating asymmetry in a changing environment: cell cycle regulation in dimorphic alphaproteobacteria.

While many bacteria divide by symmetric binary fission, some alphaproteobacteria have strikingly asymmetric cell cycles, producing offspring that differs significantly in their morphology and reproductive state. To establish this asymmetry, these species employ a complex cell cycle regulatory pathway based on two-component signaling cascades. At the center of this network is the essential DNA-binding response regulator CtrA, which acts as a transcription factor controlling numerous genes with cell cycle-relevant functions as well as a regulator of chromosome replication. The DNA-binding activity of CtrA is controlled at the level of both protein phosphorylation and stability, dependent on an intricate network of regulatory proteins, whose function is tightly coordinated in time and space. CtrA is differentially activated in the two (developing) offspring, thereby establishing distinct transcriptional programs that ultimately determine their distinct cell fates. Phase-separated polar microdomains of changing composition sequester proteins involved in the (in-)activation and degradation of CtrA specifically at each pole. In this review, we summarize the current knowledge of the CtrA pathway and discuss how it has evolved to regulate the cell cycle of morphologically distinct alphaproteobacteria.

Bacterial cell growth is arrested by violet and blue, but not yellow light excitation during fluorescence microscopy.

BACKGROUND: Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging. RESULTS: We show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445-457 nm) excitation, whereas they are largely unaffected by green light (YFP). The stress sigma factor σB and the blue-light receptor YtvA are not involved in growth arrest. Using synchronized B. subtilis cells, we show that the use of blue light for fluorescence microscopy likely induces non-specific toxic effects, rather than a specific cell cycle arrest. Escherichia coli and Caulobacter crescentus cells also stop to grow after 15 one-second exposures to blue light (CFP), but continue growth when imaged under similar conditions in the YFP channel. In the case of E. coli, YFP excitation slows growth relative to white light excitation, whereas CFP excitation leads to cell death in a majority of cells. Thus, even mild violet/blue light excitation interferes with bacterial growth. Analyzing the dose-dependent effects of violet light in B. subtilis, we show that short exposures to low-intensity violet light allow for continued cell growth, while longer exposures do not. CONCLUSIONS: Our experiments show that care must be taken in the design of live-cell imaging experiments in that violet or blue excitation effects must be closely controlled during and after imaging. Violet excitation during sptPALM or other imaging studies involving photoactivation has a threshold, below which little effects can be seen, but above which a sharp transition into cell death occurs. YFP imaging proves to be better suited for time-lapse studies, especially when cell cycle or cell growth parameters are to be examined.

Integrative and quantitative view of the CtrA regulatory network in a stalked budding bacterium.

The Alphaproteobacteria show a remarkable diversity of cell cycle-dependent developmental patterns, which are governed by the conserved CtrA pathway. Its central component CtrA is a DNA-binding response regulator that is controlled by a complex two-component signaling network, mediating distinct transcriptional programs in the two offspring. The CtrA pathway has been studied intensively and was shown to consist of an upstream part that reads out the developmental state of the cell and a downstream part that integrates the upstream signals and mediates CtrA phosphorylation. However, the role of this circuitry in bacterial diversification remains incompletely understood. We have therefore investigated CtrA regulation in the morphologically complex stalked budding alphaproteobacterium Hyphomonas neptunium. Compared to relatives dividing by binary fission, H. neptunium shows distinct changes in the role and regulation of various pathway components. Most notably, the response regulator DivK, which normally links the upstream and downstream parts of the CtrA pathway, is dispensable, while downstream components such as the pseudokinase DivL, the histidine kinase CckA, the phosphotransferase ChpT and CtrA are essential. Moreover, CckA is compartmentalized to the nascent bud without forming distinct polar complexes and CtrA is not regulated at the level of protein abundance. We show that the downstream pathway controls critical functions such as replication initiation, cell division and motility. Quantification of the signal flow through different nodes of the regulatory cascade revealed that the CtrA pathway is a leaky pipeline and must involve thus-far unidentified factors. Collectively, the quantitative system-level analysis of CtrA regulation in H. neptunium points to a considerable evolutionary plasticity of cell cycle regulation in alphaproteobacteria and leads to hypotheses that may also hold in well-established model organisms such as Caulobacter crescentus.

BacStalk: A comprehensive and interactive image analysis software tool for bacterial cell biology.

Prokaryotic cells display a striking subcellular organization. Studies of the underlying mechanisms in different species have greatly enhanced our understanding of the morphological and physiological adaptation of bacteria to different environmental niches. The image analysis software tool BacStalk is designed to extract comprehensive quantitative information from the images of morphologically complex bacteria with stalks, flagella, or other appendages. The resulting data can be visualized in interactive demographs, kymographs, cell lineage plots, and scatter plots to enable fast and thorough data analysis and representation. Notably, BacStalk can generate demographs and kymographs that display fluorescence signals within the two-dimensional cellular outlines, to accurately represent their subcellular location. Beyond organisms with visible appendages, BacStalk is also suitable for established, non-stalked model organisms with common or uncommon cell shapes. BacStalk, therefore, contributes to the advancement of prokaryotic cell biology and physiology, as it widens the spectrum of easily accessible model organisms and enables highly intuitive and interactive data analysis and visualization.

Two-step chromosome segregation in the stalked budding bacterium Hyphomonas neptunium.

Chromosome segregation typically occurs after replication has finished in eukaryotes but during replication in bacteria. Here, we show that the alphaproteobacterium Hyphomonas neptunium, which proliferates by bud formation at the tip of a stalk-like cellular extension, segregates its chromosomes in a unique two-step process. First, the two sister origin regions are targeted to opposite poles of the mother cell, driven by the ParABS partitioning system. Subsequently, once the bulk of chromosomal DNA has been replicated and the bud exceeds a certain threshold size, the cell initiates a second segregation step during which it transfers the stalk-proximal origin region through the stalk into the nascent bud compartment. Thus, while chromosome replication and segregation usually proceed concurrently in bacteria, the two processes are largely uncoupled in H. neptunium, reminiscent of eukaryotic mitosis. These results indicate that stalked budding bacteria have evolved specific mechanisms to adjust chromosome segregation to their unusual life cycle.

Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans.

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism's central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism.IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable "operating system" of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

Characterization of the first planctomycetal outer membrane protein identifies a channel in the outer membrane of the anammox bacterium Kuenenia stuttgartiensis.

Planctomycetes are a bacterial phylum known for their complex intracellular compartmentalization. While most Planctomycetes have two compartments, the anaerobic ammonium oxidizing (anammox) bacteria contain three membrane-enclosed compartments. In contrast to a long-standing consensus, recent insights suggested the outermost Planctomycete membrane to be similar to a Gram-negative outer membrane (OM). One characteristic component that differentiates OMs from cytoplasmic membranes (CMs) is the presence of outer membrane proteins (OMPs) featuring a ß-barrel structure that facilitates passage of molecules through the OM. Although proteomic and genomic evidence suggested the presence of OMPs in several Planctomycetes, no experimental verification existed of the pore-forming function and localization of these proteins in the outermost membrane of these exceptional microorganisms. Here, we show via lipid bilayer assays that at least two typical OMP-like channel-forming proteins are present in membrane preparations of the anammox bacterium Kuenenia stuttgartiensis. One of these channel-forming proteins, the highly abundant putative OMP Kustd1878, was purified to homogeneity. Analysis of the channel characteristics via lipid bilayer assays showed that Kustd1878 forms a moderately cation-selective channel with a high current noise and an average single-channel conductance of about 170-190pS in 1M KCl. Antibodies were raised against the purified protein and immunogold localization indicated Kustd1878 to be present in the outermost membrane. Therefore, this work clearly demonstrates the presence of OMPs in anammox Planctomycetes and thus firmly adds to the emerging view that Planctomycetes have a Gram-negative cell envelope.

Determinants of Bacterial Morphology: From Fundamentals to Possibilities for Antimicrobial Targeting.

Bacterial morphology is extremely diverse. Specific shapes are the consequence of adaptive pressures optimizing bacterial fitness. Shape affects critical biological functions, including nutrient acquisition, motility, dispersion, stress resistance and interactions with other organisms. Although the characteristic shape of a bacterial species remains unchanged for vast numbers of generations, periodical variations occur throughout the cell (division) and life cycles, and these variations can be influenced by environmental conditions. Bacterial morphology is ultimately dictated by the net-like peptidoglycan (PG) sacculus. The species-specific shape of the PG sacculus at any time in the cell cycle is the product of multiple determinants. Some morphological determinants act as a cytoskeleton to guide biosynthetic complexes spatiotemporally, whereas others modify the PG sacculus after biosynthesis. Accumulating evidence supports critical roles of morphogenetic processes in bacteria-host interactions, including pathogenesis. Here, we review the molecular determinants underlying morphology, discuss the evidence linking bacterial morphology to niche adaptation and pathogenesis, and examine the potential of morphological determinants as antimicrobial targets.

Determining the bacterial cell biology of Planctomycetes.

Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.

The S-Layer Protein of the Anammox Bacterium Kuenenia stuttgartiensis Is Heavily O-Glycosylated.

Anaerobic ammonium oxidation (anammox) bacteria are a distinct group of Planctomycetes that are characterized by their unique ability to perform anammox with nitrite to dinitrogen gas in a specialized organelle. The cell of anammox bacteria comprises three membrane-bound compartments and is surrounded by a two-dimensional crystalline S-layer representing the direct interaction zone of anammox bacteria with the environment. Previous results from studies with the model anammox organism Kuenenia stuttgartiensis suggested that the protein monomers building the S-layer lattice are glycosylated. In the present study, we focussed on the characterization of the S-layer protein glycosylation in order to increase our knowledge on the cell surface characteristics of anammox bacteria. Mass spectrometry (MS) analysis showed an O-glycan attached to 13 sites distributed over the entire 1591-amino acid S-layer protein. This glycan is composed of six monosaccharide residues, of which five are N-acetylhexosamine (HexNAc) residues. Four of these HexNAc residues have been identified as GalNAc. The sixth monosaccharide in the glycan is a putative dimethylated deoxyhexose. Two of the HexNAc residues were also found to contain a methyl group, thereby leading to an extensive degree of methylation of the glycan. This study presents the first characterization of a glycoprotein in a planctomycete and shows that the S-layer protein Kustd1514 of K. stuttgartiensis is heavily glycosylated with an O-linked oligosaccharide which is additionally modified by methylation. S-layer glycosylation clearly contributes to the diversification of the K. stuttgartiensis cell surface and can be expected to influence the interaction of the bacterium with other cells or abiotic surfaces.

Interfacial Activation of Candida antarctica Lipase B: Combined Evidence from Experiment and Simulation.

Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Candida antarctica lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small "lid" (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents.

Anammox Planctomycetes have a peptidoglycan cell wall.

Planctomycetes are intriguing microorganisms that apparently lack peptidoglycan, a structure that controls the shape and integrity of almost all bacterial cells. Therefore, the planctomycetal cell envelope is considered exceptional and their cell plan uniquely compartmentalized. Anaerobic ammonium-oxidizing (anammox) Planctomycetes play a key role in the global nitrogen cycle by releasing fixed nitrogen back to the atmosphere as N2. Here using a complementary array of state-of-the-art techniques including continuous culturing, cryo-transmission electron microscopy, peptidoglycan-specific probes and muropeptide analysis, we show that the anammox bacterium Kuenenia stuttgartiensis contains peptidoglycan. On the basis of the thickness, composition and location of peptidoglycan in K. stuttgartiensis, we propose to redefine Planctomycetes as Gram-negative bacteria. Our results demonstrate that Planctomycetes are not an exception to the universal presence of peptidoglycan in bacteria.

Expanding the verrucomicrobial methanotrophic world: description of three novel species of Methylacidimicrobium gen. nov.

Methanotrophic Verrucomicrobia have been found in geothermal environments characterized by high temperatures and low pH values. However, it has recently been hypothesized that methanotrophic Verrucomicrobia could be present under a broader range of environmental conditions. Here we describe the isolation and characterization of three new species of mesophilic acidophilic verrucomicrobial methanotrophs from a volcanic soil in Italy. The three new species showed 97% to 98% 16S rRNA gene identity to each other but were related only distantly (89% to 90% on the 16S rRNA level) to the thermophilic genus Methylacidiphilum. We propose the new genus Methylacidimicrobium, including the novel species Methylacidimicrobium fagopyrum, Methylacidimicrobium tartarophylax, and Methylacidimicrobium cyclopophantes. These mesophilic Methylacidimicrobium spp. were more acid tolerant than their thermophilic relatives; the most tolerant species, M. tartarophylax, still grew at pH 0.5. The variation in growth temperature optima (35 to 44°C) and maximum growth rates (µmax; 0.013 to 0.040 h(-1)) suggested that all species were adapted to a specific niche within the geothermal environment. All three species grew autotrophically using the Calvin cycle. The cells of all species contained glycogen particles and electron-dense particles in their cytoplasm as visualized by electron microscopy. In addition, the cells of one of the species (M. fagopyrum) contained intracytoplasmic membrane stacks. The discovery of these three new species and their growth characteristics expands the known diversity of verrucomicrobial methanotrophs and shows that they are present in many more ecosystems than previously assumed.

A new addition to the cell plan of anammox bacteria: "Candidatus Kuenenia stuttgartiensis" has a protein surface layer as the outermost layer of the cell.

Anammox bacteria perform anaerobic ammonium oxidation (anammox) and have a unique compartmentalized cell consisting of three membrane-bound compartments (from inside outwards): the anammoxosome, riboplasm, and paryphoplasm. The cell envelope of anammox bacteria has been proposed to deviate from typical bacterial cell envelopes by lacking both peptidoglycan and a typical outer membrane. However, the composition of the anammox cell envelope is presently unknown. Here, we investigated the outermost layer of the anammox cell and identified a proteinaceous surface layer (S-layer) (a crystalline array of protein subunits) as the outermost component of the cell envelope of the anammox bacterium "Candidatus Kuenenia stuttgartiensis." This is the first description of an S-layer in the phylum of the Planctomycetes and a new addition to the cell plan of anammox bacteria. This S-layer showed hexagonal symmetry with a unit cell consisting of six protein subunits. The enrichment of the S-layer from the cell led to a 160-kDa candidate protein, Kustd1514, which has no homology to any known protein. This protein is present in a glycosylated form. Antibodies were generated against the glycoprotein and used for immunogold localization. The antiserum localized Kustd1514 to the S-layer and thus verified that this protein forms the "Ca. Kuenenia stuttgartiensis" S-layer.

The anammoxosome organelle is crucial for the energy metabolism of anaerobic ammonium oxidizing bacteria.

Anammox bacteria convert ammonium and nitrite to dinitrogen gas under anaerobic conditions to obtain their energy for growth. The anammox reaction was deemed impossible until its discovery in the early 1990s. Now, anammox bacteria are recognized as major players in the global nitrogen cycle and estimated to be responsible for up to 50% of the nitrogen in the air that we breathe. In addition, anammox bacteria are extremely valuable for wastewater treatment where they are applied for the removal of ammonium. Besides their importance in industry and the environment, anammox bacteria defy some basic biological concepts. Whereas most other bacteria have only one cell compartment, the cytoplasm, anammox bacteria have three independent cell compartments bounded by bilayer membranes, from out- to inside; the paryphoplasm, riboplasm and anammoxosome. The anammoxosome is the largest compartment of the anammox cell and is proposed to be dedicated to energy conservation. As such it would be analogous to the mitochondria of eukaryotes. This review will discuss the anammox cell plan in detail, with the main focus on the anammoxosome. The identity of the anammoxosome as a prokaryotic organelle and the importance of this organelle for anammox bacteria are discussed as well as challenges these bacteria face by having three independent cell compartments.