Identifying components of the Shewanella phage LambdaSo lysis system.

Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase, and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization, and microscopy, we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton leakage and degradation of the cell wall. Between the corresponding genes, we found that two extensively nested open-reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR endolysin, spanin, and Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress. IMPORTANCE: Lysis of bacteria can have multiple consequences, such as the release of host DNA to foster robust biofilm. Phage-induced lysis of Gram-negative cells requires the disruption of three layers, the outer and inner membranes and the cell wall. In most cases, the lysis systems of phages infecting Gram-negative cells comprise holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membranes, completing cell envelope disruption and allowing phage egress. Here, we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages, further factors may be required.

Daptomycin-Impregnated PMMA Cement against Vancomycin-Resistant Germs: Dosage, Handling, Elution, Mechanical Stability, and Effectiveness.

BACKGROUND: The number of periprosthetic joint infections caused by vancomycin-resistant pathogens is increasing. Currently, no PMMA cement is commercially available to cover VRE. Daptomycin shows promising results in treating infection, offering a good safety profile and a reduced risk of developing resistance. The purpose of this in vitro study was to investigate the mechanical stability, handling properties, elution behavior, and antimicrobial effectiveness of PMMA cement loaded with three different daptomycin concentrations in comparison to commercially available antibiotic-loaded bone cement (ALBC). METHODS: Mechanical properties and handling characteristics (ISO 5833, DIN 53435), HPLC elution, antimicrobial effectiveness with proliferation assay (DIN 17025), and inhibition zone testing were investigated. RESULTS: All tested daptomycin concentrations met the ISO and DIN standards for mechanical strength. Loading of 40 g of PMMA cement with 0.5 g of daptomycin did not show any antimicrobial effectiveness, in contrast to 1.0 g and 1.5 g. PMMA cement with 1.5 g of daptomycin was the best in terms of elution and effectiveness, and it showed good ISO mechanical strength; ISO doughing was sticky for a little longer and setting was faster compared to the vancomycin-containing reference cement. CONCLUSION: PMMA cement containing 0.5 g of gentamicin and 1.5 g of daptomycin could be a good alternative to the already established COPAL® (Wehrheim, Germany) G+V for the treatment of PJIs caused by VRE.

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

Polarity of c-di-GMP synthesis and degradation.

The bacterial cell pole has long been recognized as a defined compartment for enzymatic activities that are important or even vital for the cell. Polarity of diguanylate cyclases and phosphodiesterases, enzymes that synthesize and degrade the second messenger c-di-GMP, has now been demonstrated for several bacterial systems. Here we review these polar regulatory systems and show how the asymmetry of c-di-GMP production and turnover in concert with different modes of activation and deactivation creates heterogeneity in cellular c-di-GMP levels. We highlight how this heterogeneity generates a diverse set of phenotypic identities or states and how this may benefit the cell population, and we discuss reasons why the polarity of c-di-GMP signaling is probably widespread among bacteria.

FlrA-independent production of flagellar proteins is required for proper flagellation in Shewanella putrefaciens.

Flagella are multiprotein complexes whose assembly and positioning require complex spatiotemporal control. Flagellar assembly is thought to be controlled by several transcriptional tiers, which are mediated through various master regulators. Here, we revisited the regulation of flagellar genes in polarly flagellated gammaproteobacteria by the regulators FlrA, RpoN (σ54 ) and FliA (σ28 ) in Shewanella putrefaciens CN-32 at the transcript and protein level. We found that a number of regulatory and structural proteins were present in the absence of the main regulators, suggesting that initiation of flagella assembly and motor activation relies on the abundance control of only a few structural key components that are required for the formation of the MS- and C-ring and the flagellar type III secretion system. We identified FlrA-independent promoters driving expression of the regulators of flagellar number and positioning, FlhF and FlhG. Reduction of the gene expression levels from these promoters resulted in the emergence of hyperflagellation. This finding indicates that basal expression is required to adjust the flagellar counter in Shewanella. This is adding a deeper layer to the regulation of flagellar synthesis and assembly.

Wrapped Up: The Motility of Polarly Flagellated Bacteria.

A huge number of bacterial species are motile by flagella, which allow them to actively move toward favorable environments and away from hazardous areas and to conquer new habitats. The general perception of flagellum-mediated movement and chemotaxis is dominated by the Escherichia coli paradigm, with its peritrichous flagellation and its famous run-and-tumble navigation pattern, which has shaped the view on how bacteria swim and navigate in chemical gradients. However, a significant amount-more likely the majority-of bacterial species exhibit a (bi)polar flagellar localization pattern instead of lateral flagella. Accordingly, these species have evolved very different mechanisms for navigation and chemotaxis. Here, we review the earlier and recent findings on the various modes of motility mediated by polar flagella.

Polar flagellar wrapping and lateral flagella jointly contribute to Shewanella putrefaciens environmental spreading.

Flagella enable bacteria to actively spread within the environment. A number of species possess two separate flagellar systems, where in most cases a primary polar flagellar system is supported by distinct secondary lateral flagella under appropriate conditions. Using functional fluorescence tagging on one of these species, Shewanella putrefaciens, as a model system, we explored how two different flagellar systems can exhibit efficient joint function. The S. putrefaciens secondary flagellar filaments are composed as a mixture of two highly homologous non-glycosylated flagellins, FlaA2 and FlaB2 . Both are solely sufficient to form a functional filament, however, full spreading motility through soft agar requires both flagellins. During swimming, lateral flagella emerge from the cell surface at angles between 30° and 50°, and only filaments located close to the cell pole may form a bundle. Upon a directional shift from forward to backward swimming initiated by the main polar flagellum, the secondary filaments flip over and thus support propulsion into either direction. Lateral flagella do not inhibit the wrapping of the polar flagellum around the cell body at high load. Accordingly, screw thread-like motility mediated by the primary flagellum and activity of lateral flagella cumulatively supports spreading through constricted environments such as polysaccharide matrices.

GGDEF domain as spatial on-switch for a phosphodiesterase by interaction with landmark protein HubP.

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bimodal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here, we show that direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the FimV domain of the polar landmark protein HubP is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface-adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations, generated by differences in abundance and timing of polar appearance of PdeB, orchestrates the population behavior with respect to cell-surface interaction and environmental spreading.

Dynamic Hybrid Flagellar Motors-Fuel Switch and More.

Flagellar motors are intricate rotating nanomachines that are powered by transmembrane ion gradients. The stator complexes are the powerhouses of the flagellar motor: They convert a transmembrane ion gradient, mainly of H+ or Na+, into rotation of the helical flagellar filament. They are thus essential for motor function. The number of stators synchronously engaged in the motor is surprisingly dynamic and depends on the load and the environmental concentration of the corresponding coupling ion. Thus, the rotor-stator interactions determine an important part of the properties of the motor. Numerous bacteria have been identified as possessing more than one set of stators, and some species have been demonstrated to use these different stators in various configurations to modify motor functions by dynamic in-flight swapping. Here, we review knowledge of the properties, the functions, and the evolution of these hybrid motors and discuss questions that remain unsolved.

Corrigendum: The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

[This corrects the article DOI: 10.3389/fmicb.2021.668892.].

Obligate cross-feeding expands the metabolic niche of bacteria.

Bacteria frequently engage in obligate metabolic mutualisms with other microorganisms. However, it remains generally unclear how the resulting metabolic dependencies affect the ecological niche space accessible to the whole consortium relative to the niche space available to its constituent individuals. Here we address this issue by systematically cultivating metabolically dependent strains of different bacterial species either individually or as pairwise cocultures in a wide range of carbon sources. Our results show that obligate cross-feeding is significantly more likely to expand the metabolic niche space of interacting bacterial populations than to contract it. Moreover, niche expansion occurred predominantly between two specialist taxa and correlated positively with the phylogenetic distance between interaction partners. Together, our results demonstrate that obligate cross-feeding can significantly expand the ecological niche space of interacting bacterial genotypes, thus explaining the widespread occurrence of this type of ecological interaction in natural microbiomes.

The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

A number of bacterial species control the function of the flagellar motor in response to the levels of the secondary messenger c-di-GMP, which is often mediated by c-di-GMP-binding proteins that act as molecular brakes or clutches to slow the motor rotation. The gammaproteobacterium Shewanella putrefaciens possesses two distinct flagellar systems, the primary single polar flagellum and a secondary system with one to five lateral flagellar filaments. Here, we identified a protein, MotL, which specifically regulates the activity of the lateral, but not the polar, flagellar motors in response to the c-di-GMP levels. MotL only consists of a single PilZ domain binding c-di-GMP, which is crucial for its function. Deletion and overproduction analyses revealed that MotL slows down the lateral flagella at elevated levels of c-di-GMP, and may speed up the lateral flagellar-mediated movement at low c-di-GMP concentrations. In vitro interaction studies hint at an interaction of MotL with the C-ring of the lateral flagellar motors. This study shows a differential c-di-GMP-dependent regulation of the two flagellar systems in a single species, and implicates that PilZ domain-only proteins can also act as molecular regulators to control the flagella-mediated motility in bacteria.

Dynamics of Bacterial Signal Recognition Particle at a Single Molecule Level.

We have studied the localization and dynamics of bacterial Ffh, part of the SRP complex, its receptor FtsY, and of ribosomes in the Gamma-proteobacterium Shewanella putrefaciens. Using structured illumination microscopy, we show that ribosomes show a pronounced accumulation at the cell poles, whereas SRP and FtsY are distributed at distinct sites along the cell membrane, but they are not accumulated at the poles. Single molecule dynamics can be explained by assuming that all three proteins/complexes move as three distinguishable mobility fractions: a low mobility/static fraction may be engaged in translation, medium-fast diffusing fractions may be transition states, and high mobility populations likely represent freely diffusing molecules/complexes. Diffusion constants suggest that SRP and FtsY move together with slow-mobile ribosomes. Inhibition of transcription leads to loss of static molecules and reduction of medium-mobile fractions, in favor of freely diffusing subunits, while inhibition of translation appears to stall the medium mobile fractions. Depletion of FtsY leads to aggregation of Ffh, but not to loss of the medium mobile fraction, indicating that Ffh/SRP can bind to ribosomes independently from FtsY. Heat maps visualizing the three distinct diffusive populations show that while static molecules are mostly clustered at the cell membrane, diffusive molecules are localized throughout the cytosol. The medium fast populations show an intermediate pattern of preferential localization, suggesting that SRP/FtsY/ribosome transition states may form within the cytosol to finally find a translocon.

Antibiotic Drug screening and Image Characterization Toolbox (A.D.I.C.T.): a robust imaging workflow to monitor antibiotic stress response in bacterial cells in vivo.

The search for novel drugs that efficiently eliminate prokaryotic pathogens is one of the most urgent health topics of our time. Robust evaluation methods for monitoring the antibiotic stress response in prokaryotes are therefore necessary for developing respective screening strategies. Besides advantages of common in vitro techniques, there is a growing demand for in vivo information based on imaging techniques that allow to screen antibiotic candidates in a dynamic manner. Gathering information from imaging data in a reproducible manner, robust data processing and analysis workflows demand advanced (semi-)automation and data management to increase reproducibility. Here we demonstrate a versatile and robust semi-automated image acquisition, processing and analysis workflow to investigate bacterial cell morphology in a quantitative manner. The presented workflow, A.D.I.C.T, covers aspects of experimental setup deployment, data acquisition and handling, image processing (e.g. ROI management, data transformation into binary images, background subtraction, filtering, projections) as well as statistical evaluation of the cellular stress response (e.g. shape measurement distributions, cell shape modeling, probability density evaluation of fluorescence imaging micrographs) towards antibiotic-induced stress, obtained from time-course experiments. The imaging workflow is based on regular brightfield images combined with live-cell imaging data gathered from bacteria, in our case from recombinant Shewanella cells, which are processed as binary images. The model organism expresses target proteins relevant for membrane-biogenesis that are functionally fused to respective fluorescent proteins. Data processing and analysis are based on customized scripts using ImageJ2/FIJI, Celltool and R packages that can be easily reproduced and adapted by users. Summing up, our approach aims at supporting life-scientists to establish their own imaging-pipeline in order to exploit their data as versatile as possible and in a reproducible manner.

Isolation and Characterization of Shewanella Phage Thanatos Infecting and Lysing Shewanella oneidensis and Promoting Nascent Biofilm Formation.

Species of the genus Shewanella are widespread in nature in various habitats, however, little is known about phages affecting Shewanella sp. Here, we report the isolation of phages from diverse freshwater environments that infect and lyse strains of Shewanella oneidensis and other Shewanella sp. Sequence analysis and microscopic imaging strongly indicate that these phages form a so far unclassified genus, now named Shewanella phage Thanatos, which can be positioned within the subfamily of Tevenvirinae (Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Caudovirales; Myoviridae; Tevenvirinae). We characterized one member of this group in more detail using S. oneidensis MR-1 as a host. Shewanella phage Thanatos-1 possesses a prolate icosahedral capsule of about 110 nm in height and 70 nm in width and a tail of about 95 nm in length. The dsDNA genome exhibits a GC content of about 34.5%, has a size of 160.6 kbp and encodes about 206 proteins (92 with an annotated putative function) and two tRNAs. Out of those 206, MS analyses identified about 155 phage proteins in PEG-precipitated samples of infected cells. Phage attachment likely requires the outer lipopolysaccharide of S. oneidensis, narrowing the phage's host range. Under the applied conditions, about 20 novel phage particles per cell were produced after a latent period of approximately 40 min, which are stable at a pH range from 4 to 12 and resist temperatures up to 55°C for at least 24 h. Addition of Thanatos to S. oneidensis results in partial dissolution of established biofilms, however, early exposure of planktonic cells to Thanatos significantly enhances biofilm formation. Taken together, we identified a novel genus of Myophages affecting S. oneidensis communities in different ways.

An ATP-dependent partner switch links flagellar C-ring assembly with gene expression.

Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.

A Proline-Rich Element in the Type III Secretion Protein FlhB Contributes to Flagellar Biogenesis in the Beta- and Gamma-Proteobacteria.

Flagella are bacterial organelles of locomotion. Their biogenesis is highly coordinated in time and space and relies on a specialized flagellar type III secretion system (fT3SS) required for the assembly of the extracellular hook, rod, and filament parts of this complex motor device. The fT3SS protein FlhB switches secretion substrate specificity once the growing hook reaches its determined length. Here we present the crystal structure of the cytoplasmic domain of the transmembrane protein FlhB. The structure visualizes a so-far unseen proline-rich region (PRR) at the very C-terminus of the protein. Strains lacking the PRR show a decrease in flagellation as determined by hook- and filament staining, indicating a role of the PRR during assembly of the hook and filament structures. Phylogenetic analysis shows that the PRR is a primary feature of FlhB proteins of flagellated beta- and gamma-proteobacteria. Taken together, our study adds another layer of complexity and organismic diversity to the process of flagella biogenesis.

NosP Signaling Modulates the NO/H-NOX-Mediated Multicomponent c-Di-GMP Network and Biofilm Formation in Shewanella oneidensis.

Biofilms form when bacteria aggregate in a self-secreted exopolysaccharide matrix; they are resistant to antibiotics and implicated in disease. Nitric oxide (NO) is known to mediate biofilm formation in many bacteria via ligation to H-NOX (heme-NO/oxygen binding) domains. Most NO-responsive bacteria, however, lack H-NOX domain-containing proteins. We have identified another NO-sensing protein (NosP), which is predicted to be involved in two-component signaling and biofilm regulation in many species. Here, we demonstrate that NosP participates in the previously described H-NOX/NO-responsive multicomponent c-di-GMP signaling network in Shewanella oneidensis. Strains lacking either nosP or its co-cistronic kinase nahK (previously hnoS) produce immature biofilms, while hnoX and hnoK (kinase responsive to NO/H-NOX) mutants result in wild-type biofilm architecture. We demonstrate that NosP regulates the autophosphorylation activity of NahK as well as HnoK. HnoK and NahK have been shown to regulate three response regulators (HnoB, HnoC, and HnoD) that together comprise a NO-responsive multicomponent c-di-GMP signaling network. Here, we propose that NosP/NahK adds regulation on top of H-NOX/HnoK to modulate this c-di-GMP signaling network, and ultimately biofilm formation, by governing the flux of phosphate through both HnoK and NahK. In addition, it appears that NosP and H-NOX act to counter each other in a push-pull mechanism; NosP/NahK promotes biofilm formation through inhibition of H-NOX/HnoK signaling, which itself reduces the extent of biofilm formation. Addition of NO results in a reduction of c-di-GMP and biofilm formation, primarily through disinhibition of HnoK activity.

Breakdown of Vibrio cholerae biofilm architecture induced by antibiotics disrupts community barrier function.

Bacterial cells in nature are frequently exposed to changes in their chemical environment1,2. The response mechanisms of isolated cells to such stimuli have been investigated in great detail. By contrast, little is known about the emergent multicellular responses to environmental changes, such as antibiotic exposure3-7, which may hold the key to understanding the structure and functions of the most common type of bacterial communities: biofilms. Here, by monitoring all individual cells in Vibrio cholerae biofilms during exposure to antibiotics that are commonly administered for cholera infections, we found that translational inhibitors cause strong effects on cell size and shape, as well as biofilm architectural properties. We identified that single-cell-level responses result from the metabolic consequences of inhibition of protein synthesis and that the community-level responses result from an interplay of matrix composition, matrix dissociation and mechanical interactions between cells. We further observed that the antibiotic-induced changes in biofilm architecture have substantial effects on biofilm population dynamics and community assembly by enabling invasion of biofilms by bacteriophages and intruder cells of different species. These mechanistic causes and ecological consequences of biofilm exposure to antibiotics are an important step towards understanding collective bacterial responses to environmental changes, with implications for the effects of antimicrobial therapy on the ecological succession of biofilm communities.