High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells.

Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.

Studies on the enzymatic specificity of diguanylate cyclases domains and the production of new second messengers / Estudos na especificidade enzimática de diguanilato ciclases e na produção de novos segundos mensageiros

In the first chapter, studies on substrate recognition and enzymatic activity of GGDEF domains are presented. Many proteins containing GGDEF domains are diguanylate cyclases (DGCs, EC 2.7.7.65), enzymes that catalyze the conversion of 2 GTP molecules into the second messenger c-di-GMP in prokaryotes. This molecule is primarily implicated in the transition between motile and sessile lifestyles, as well several other phenotypes. Redundancy and diversity of GGDEF domain sequences in many bacterial genomes raises the possibility that other enzymatic functions may yet be discovered. To test this hypothesis, i) the effect of point mutations on the structure and enzymatic activity of GGDEF domains is analyzed, ii) the enzymatic specificity of wild-type GGDEF domains from different proteins is also tested, and iii) when non-canonical products are detected, enzymatic models are studied to understand its preferential production. The principal results obtained from these studies are as follows. Seven mutants of the DGC PleD (a GGDEF containing-protein from Caulobacter crescentus) were constructed and the crystallographic structure of two of them was solved, showing that they are unlikely to bind the guanine moiety in its active site. Additionally, five mutants of XAC0610, another DGC from Xanthomonas citr, were constructed and their substrate specificities were evaluated. None of those mutants were able to use ATP as a substrate. Finally, seven different GGDEF domain-containing DGCs from different sources were expressed and purified and their enzymatic specificities were tested with several nucleotide triphosphates. One enzyme, GSU1658 from Geobacter sulfurreducens was particularly promiscuous and shown to produce c-di-GMP, c-di-AMP, c-di-IMP, c-di-2´dGMP, cGAMP, c-GIMP, and c-AIMP. Interestingly, XAC0610 was able to recognize 2´dGTP as substrate. Analysis of enzyme kinetics of XAC0610 in presence of 2´dGTP and/or GTP showed the preferential formation of the hybrid linear product pppGp2´dG. The second chapter present studies on cyanide metabolism in Bacillus with focus on the cyanide dihydratase of Bacillus safensis. Cyanide is widely used in industries due to its high affinity for metals. This same ability confers potent toxicity to this compound. Thus, industries must reduce the cyanide concentration from wastewater before its final disposal. Physical, chemical, and biological methods have been developed to achieve this goal, but knowledge about metabolic pathways and the biology of enzymes involved in cyanide degradation is still scarce. Here, the isolation of a Bacillus safensis strain from mine tailings in Peru is described. Classification of this strain was done through a comparative analysis of 132 core genomes of strains from the Bacillus pumilus group. Sequence analysis determined that a cyanide dihydratase (CynD, EC 3.5.5.1)) encoded in the genome of the isolated strain was likely the enzyme responsible for cyanide degradation. Confirmation of the cyanide degrading activity of CynD from this strain was achieved by cloning, expression and purification of the enzyme and its enzymatic characterization. CynD from this strain was active up to pH 9 and oligomerization patterns analyzed by SEC-MALS and electron microscopy showed that the enzyme forms large helical structures at pH 8 and smaller structures at higher pHs. Finally, we show that CynD expression is strongly induced in the presence of cyanide. The last two years of graduate studies were carried out in the context of the COVID-19 pandemic. Thanks to the large amount of publicly available genomic data, we were able to carry out studies on the worldwide dynamics of the spread of SARS-CoV-2 mutants forms. In the first year of the pandemic, genomic classification of 171,461 genomes showed the presence of five major haplotypes based on nine mutations. The worldwide distribution and the temporal evolution of frequency of these haplotypes was carefully analyzed. All the haplotypes were identified in the six regions analyzed (South America, North America, Europe, Asia, Africa, and Oceania); however, the frequency of each of them was different in each of these regions. As of September 30, 2020, haplotype 3 (or operational taxonomic unit 3, OTU_3) was the most prevalent in four regions (South America, Asia, Africa, and Oceania). OTU_5 was the most prevalent in North America and OTU_2 in Europe. Temporal dynamics of the haplotypes showed that OTU_1 became nearly extinct after 8 months of pandemic (November 2020). Other OTUs are still present in different frequencies all around the world, while currently generating new variants. Based on their temporal dynamics, a classification scheme of 115 SARS-CoV-2 mutations identified from 1,058,020 SARS-COV-2 genomes was also performed. Three types of temporal dynamics of mutations were identified: i) High-Frequency mutations are characterized by a rapid increase in frequency upon its appearance, ii) medium and iii) low-frequency mutations maintain mid or low-frequencies for several months and can be region-specific. Finally, we performed a correlation analysis of the effective reproduction number (Rt) of SARS-CoV-2 harboring the high-frequency mutation N501Y with the level of control measures adopted in specific jurisdictions. We show that Rt is negatively correlated with the level of control measures in eight of the nine countries analyzed. This negative correlation was similar when we analyzed the Rt of SARS-CoV-2 not-harboring N501Y. Thus, the control measures likely diminish the Rt of both SARSCoV-2 wild-type and N501Y

O presente trabalho está dividido em três capítulos sobre linhas de pesquisa diferentes desenvolvidas pelo autor durante o período de doutorado No primeiro capítulo, são apresentados estudos relacionados ao reconhecimento estrutural de substratos e análise enzimática de domínios GGDEF com atividade diguanilato ciclase (EC 2.7.7.65). As proteínas contendo domínios GGDEF estão relacionados à produção enzimática do segundo mensageiro c-di-GMP, a partir de duas moléculas de GTP, em procariotos. Esta molécula está principalmente envolvida na transição entre os estilos de vida móveis e sésseis, bem como vários outros fenótipos. Redundância e diversidade de sequências de domínio GGDEF aumentam a possibilidade de que outras funções enzimáticas ainda possam ser descobertas. Para testar esta hipótese, i) o efeito de mutações pontuais na estrutura e atividade enzimática dos domínios GGDEF é analisado, ii) a especificidade enzimática de domínios GGDEF de enzimas diferentes também é testada e iii) quando produtos não canônicos são detectados, modelos enzimáticos são estudados para entender sua produção preferencial. Como resultados mais importantes, sete mutantes do PleD (uma proteína contendo GGDEF) foram construídos e a estrutura cristalográfica de dois delas foi resolvida, mostrando que é improvável que eles liguem à porção guanina em seu sítio ativo. Além disso, cinco mutantes da proteína XAC0610 de Xanthomonas citri foram construídos e sua capacidade de usar ATP ou GTP como substrato foi avaliada. Nenhum desses mutantes foi capaz de usar ATP como substrato. Finalmente, sete outras proteínas contendo GGDEF foram purificadas e sua especificidade enzimática foi avaliada com vários trifosfatos de nucleotídeos. Uma enzima promíscua chamada GSU1658 mostrou produzir c-di-GMP, c-di-AMP, c-di-IMP, c-di-2´dGMP, c-GAMP, cGIMP e c-AIMP. Curiosamente, o XAC0610 foi capaz de reconhecer 2´dGTP como substrato. A análise da cinética enzimática de XAC0610 na presença de 2´dGTP e GTP mostrou a formação preferencial do produto linear híbrido pppGp2´dG. O segundo capítulo aborda estudos sobre o metabolismo do cianeto em Bacillus com foco na cianeto dihidratase de Bacillus safensis. O cianeto é amplamente utilizado nas indústrias devido à sua alta afinidade com os metais. Esta mesma capacidade confere toxicidade potente a este composto. Assim, as indústrias têm que reduzir a concentração de cianeto das águas residuais antes de sua disposição final. Métodos físicos, químicos e biológicos têm sido desenvolvidos para atingir esse objetivo, mas o conhecimento sobre as vias metabólicas e a biologia das enzimas envolvidas na degradação do cianeto ainda é escasso. Aqui, é descrito o isolamento de uma cepa de Bacillus safensis de rejeitos de minas no Peru. A classificação desta cepa foi feita através de uma análise comparativa de 132 core genomes de cepas do grupo de Bacillus pumilus. Em seguida, determinamos que uma cianeto dihidratase (CynD, EC 3.5.5.1) codificada no genoma da cepa isolada era provavelmente a enzima responsável pela degradação do cianeto. A confirmação da atividade degradante de cianeto de CynD desta cepa foi feita por clonagem, expressão e purificação da enzima e realização de caracterização enzimática. O CynD desta cepa é ativo até pH 9 e os padrões de oligomerização analisados por SEC-MALS mostraram que a enzima forma longas estruturas helicoidais em pH 8 e estruturas menores enquanto o pH aumenta. Finalmente, foi demonstrado que a expressão de CynD é fortemente induzida na presença de cianeto. Os últimos dois anos do doutorado foram realizados no contexto da pandemia COVID- 19. Vários laboratórios se dedicaram a gerar conhecimento para ajudar no combate à pandemia. Nesta situação e graças à grande quantidade de dados genômicos disponíveis publicamente, estudos sobre a dinâmica das mutações do SARS-CoV-2 foram realizados. No primeiro ano da pandemia, a classificação genômica de 171.461 genomas mostrou a presença de cinco haplótipos principais com base em nove mutações. A distribuição mundial e a mudança de frequência desses haplótipos foram analisadas cuidadosamente. Todos os haplótipos foram identificados nas seis regiões analisadas (América do Sul, América do Norte, Europa, Ásia, África e Oceania); no entanto, a frequência de cada um deles foi diferente em cada uma dessas regiões. Em 30 de setembro de 2020, o haplótipo 3 (ou unidade taxonômica operacional 3, OTU_3) era o mais prevalente em quatro regiões (América do Sul, Ásia, África e Oceania). OTU_5 foi o mais prevalente na América do Norte e OTU_2 na Europa. A dinâmica temporal dos haplótipos mostrou que OTU_1 parece perto da extinção após 8 meses de pandemia (novembro de 2020). Outros OTUs ainda estão presentes em diferentes frequências em todo o mundo, mesmo atualmente gerando novas variantes. Com base em sua dinâmica temporal, um esquema de classificação de 115 mutações SARS-CoV-2 identificadas a partir de 1.058.020 genomas SARS-COV-2 também foi feito. Três tipos de dinâmica temporal de mutações foram identificados: i) Mutações de alta frequência, ii) mutações de média frequência e iii) mutações de baixa frequência. Finalmente, foi analisada a correlação do número de reprodução efetiva (Rt) do SARS-CoV-2 que contém a mutação de alta frequência N501Y com o nível de medidas de controle, mostrando que seu Rt está negativamente correlacionado com o nível de medidas de controle em oito dos nove países analisados. Esta correlação negativa foi semelhante quando foi analisado o Rt de SARS-CoV-2 sem a mutação N501Y. Assim, as medidas de controle provavelmente diminuirão o Rt de SARS-CoV-2 tipo selvagem e N501Y

The DNA Recognition Motif of GapR Has an Intrinsic DNA Binding Preference towards AT-rich DNA.

The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3' end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1-72, GapRΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions.

Cold Regulation of Genes Encoding Ion Transport Systems in the Oligotrophic Bacterium Caulobacter crescentus.

In this study, we characterize the response of the free-living oligotrophic alphaproteobacterium Caulobacter crescentus to low temperatures by global transcriptomic analysis. Our results showed that 656 genes were upregulated and 619 were downregulated at least 2-fold after a temperature downshift. The identified differentially expressed genes (DEG) belong to several functional categories, notably inorganic ion transport and metabolism, and a subset of these genes had their expression confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Several genes belonging to the ferric uptake regulator (Fur) regulon were downregulated, indicating that iron homeostasis is relevant for adaptation to cold. Several upregulated genes encode proteins that interact with nucleic acids, particularly RNA: cspA, cspB, and the DEAD box RNA helicases rhlE, dbpA, and rhlB. Moreover, 31 small regulatory RNAs (sRNAs), including the cell cycle-regulated noncoding RNA (ncRNA) CcnA, were upregulated, indicating that posttranscriptional regulation is important for the cold stress response. Interestingly, several genes related to transport were upregulated under cold stress, including three AcrB-like cation/multidrug efflux pumps, the nitrate/nitrite transport system, and the potassium transport genes kdpFABC. Further characterization showed that kdpA is upregulated in a potassium-limited medium and at a low temperature in a SigT-independent way. kdpA mRNA is less stable in rho and rhlE mutant strains, but while the expression is positively regulated by RhlE, it is negatively regulated by Rho. A kdpA-deleted strain was generated, and its viability in response to osmotic, acidic, or cold stresses was determined. The implications of such variation in the gene expression for cold adaptation are discussed. IMPORTANCE Low-temperature stress is an important factor for nucleic acid stability and must be circumvented in order to maintain the basic cell processes, such as transcription and translation. The oligotrophic lifestyle presents further challenges to ensure the proper nutrient uptake and osmotic balance in an environment of slow nutrient flow. Here, we show that in Caulobacter crescentus, the expression of the genes involved in cation transport and homeostasis is altered in response to cold, which could lead to a decrease in iron uptake and an increase in nitrogen and high-affinity potassium transport by the Kdp system. This previously uncharacterized regulation of the Kdp transporter has revealed a new mechanism for adaptation to low temperatures that may be relevant for oligotrophic bacteria.

The Chaperonin GroESL Facilitates Caulobacter crescentus Cell Division by Supporting the Functions of the Z-Ring Regulators FtsA and FzlA.

The highly conserved chaperonin GroESL performs a crucial role in protein folding; however, the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events, such as DNA replication and chromosome segregation, are able to continue when GroESL folding is insufficient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide.IMPORTANCE All organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and the cell division proteins FzlA and FtsA, which modulate Z-ring function. FtsA is a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

vLUME: 3D virtual reality for single-molecule localization microscopy.

vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.

Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS.

Technical developments are unifying molecular and cellular biology. A recent electron cryotomography study by von Kügelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.

Chiral twisting in a bacterial cytoskeletal polymer affects filament size and orientation.

In many rod-shaped bacteria, the actin homolog MreB directs cell-wall insertion and maintains cell shape, but it remains unclear how structural changes to MreB affect its organization in vivo. Here, we perform molecular dynamics simulations for Caulobacter crescentus MreB to extract mechanical parameters for inputs into a coarse-grained biophysical polymer model that successfully predicts MreB filament properties in vivo. Our analyses indicate that MreB double protofilaments can exhibit left-handed twisting that is dependent on the bound nucleotide and membrane binding; the degree of twisting correlates with the length and orientation of MreB filaments observed in vitro and in vivo. Our molecular dynamics simulations also suggest that membrane binding of MreB double protofilaments induces a stable membrane curvature of similar magnitude to that observed in vivo. Thus, our multiscale modeling correlates cytoskeletal filament size with conformational changes inferred from molecular dynamics simulations, providing a paradigm for connecting protein filament structure and mechanics to cellular organization and function.

The structure of bactofilin filaments reveals their mode of membrane binding and lack of polarity.

Bactofilins are small ß-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions, from cell stalk formation in Caulobacter crescentus to chromosome segregation and motility in Myxococcus xanthus. However, the precise molecular architecture of bactofilin filaments has remained unclear. Here, sequence analysis and electron microscopy results reveal that, in addition to being widely distributed across bacteria and archaea, bactofilins are also present in a few eukaryotic lineages such as the Oomycetes. Electron cryomicroscopy analysis demonstrated that the sole bactofilin from Thermus thermophilus (TtBac) forms constitutive filaments that polymerize through end-to-end association of the ß-helical domains. Using a nanobody, we determined the near-atomic filament structure, showing that the filaments are non-polar. A polymerization-impairing mutation enabled crystallization and structure determination, while reaffirming the lack of polarity and the strength of the ß-stacking interface. To confirm the generality of the lack of polarity, we performed coevolutionary analysis on a large set of sequences. Finally, we determined that the widely conserved N-terminal disordered tail of TtBac is responsible for direct binding to lipid membranes, both on liposomes and in Escherichia coli cells. Membrane binding is probably a common feature of these widespread but only recently discovered filaments of the prokaryotic cytoskeleton.

Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly.

Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C. crescentus, we show that 2D protein self-assembly is sufficient to build and maintain the S-layer in living cells by efficient protein crystal nucleation and growth. We propose a model supported by single-molecule tracking whereby randomly secreted SLP monomers diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated at the edges of growing 2D S-layer crystals. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Unsupervised assembly poses challenges for therapeutics targeting S-layers. However, protein crystallization as an evolutionary driver rationalizes S-layer diversity and raises the potential for biologically inspired self-assembling macromolecular nanomaterials.

Caulobacter crescentus Hfq structure reveals a conserved mechanism of RNA annealing regulation.

We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.

Applicability of Instability Index for In vitro Protein Stability Prediction.

BACKGROUND: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. OBJECTIVE: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. METHODS: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-ß-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. RESULTS: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. CONCLUSION: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

Bacterial cellobiose metabolism: An inquiry-driven, comprehensive undergraduate laboratory teaching approach to promote investigative learning.

Technique-centered biochemistry or molecular biology undergraduate laboratory curricula do not offer significant opportunities for thoughtful, in-depth exploration of the science to foster investigative learning. To demonstrate inclusion of inquiry-driven laboratory experiments into the undergraduate biochemistry and molecular biology curricula, a comprehensive set of laboratory experiments, covering several principles of biochemistry and molecular biology, have been developed under a single theme. The laboratory curriculum described here comprehensively investigates bacterial cellobiose metabolism using multiple biochemical, molecular biological (RNA isolation, RT-PCR, PCR, and enzyme assay), and analytical techniques (High Performance Liquid Chromatography, NMR, spectrophotometry, and thin-layer chromatography) to explore the principles of metabolomics and genomics in a single undergraduate laboratory course setting using Caulobacter crescentus as the model organism. This laboratory module serves as a model for educators to develop easy-to-implement laboratory curricula incorporating contemporary biochemistry and molecular biology concepts and techniques to provide a course-based undergraduate research experiences (CUREs) with defined learning objectives. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):438-445, 2019.

Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus.

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod-shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme-resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD-crosslinks between meso-diaminopimelic acid and D-alanine residues, whereas stalk peptidoglycan had more LD-transpeptidation (meso-diaminopimelic acid-meso-diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD-transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD-crosslinking. Despite no obvious selection pressure for LD-crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP-DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.

Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging.

Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.

Integration of cell cycle signals by multi-PAS domain kinases.

Spatial control of intracellular signaling relies on signaling proteins sensing their subcellular environment. In many cases, a large number of upstream signals are funneled to a master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly integrate a complex array of signals within the appropriate spatial niche within the cell. As a model for how subcellular spatial information can control signaling activity, we have reconstituted the cell pole-specific control of the master regulator kinase/phosphatase CckA from the asymmetrically dividing bacterium Caulobacter crescentus CckA is active as a kinase only when it accumulates within a microdomain at the new cell pole, where it colocalizes with the pseudokinase DivL. Both proteins contain multiple PAS domains, a multifunctional class of sensory domains present across the kingdoms of life. Here, we show that CckA uses its PAS domains to integrate information from DivL and its own oligomerization state to control the balance of its kinase and phosphatase activities. We reconstituted the DivL-CckA complex on liposomes in vitro and found that DivL directly controls the CckA kinase/phosphatase switch, and that stimulation of either CckA catalytic activity depends on the second of its two PAS domains. We further show that CckA oligomerizes through a multidomain interaction that is critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase activity is independent of CckA homooligomerization. Our results broadly demonstrate how signaling factors can leverage information from their subcellular niche to drive spatiotemporal control of cell signaling.

Cyclic di-GMP differentially tunes a bacterial flagellar motor through a novel class of CheY-like regulators.

The flagellar motor is a sophisticated rotary machine facilitating locomotion and signal transduction. Owing to its important role in bacterial behavior, its assembly and activity are tightly regulated. For example, chemotaxis relies on a sensory pathway coupling chemical information to rotational bias of the motor through phosphorylation of the motor switch protein CheY. Using a chemical proteomics approach, we identified a novel family of CheY-like (Cle) proteins in Caulobacter crescentus, which tune flagellar activity in response to binding of the second messenger c-di-GMP to a C-terminal extension. In their c-di-GMP bound conformation Cle proteins interact with the flagellar switch to control motor activity. We show that individual Cle proteins have adopted discrete cellular functions by interfering with chemotaxis and by promoting rapid surface attachment of motile cells. This study broadens the regulatory versatility of bacterial motors and unfolds mechanisms that tie motor activity to mechanical cues and bacterial surface adaptation.

Mathematical modeling of spatiotemporal protein localization patterns in C. crescentus bacteria: A mechanism for asymmetric FtsZ ring positioning.

We examine the localization patterns of ParA, ParB, PopZ, and MipZ, which are key division proteins in C. crescentus bacteria. While Par and PopZ proteins have been implicated in the physical segregation of the replicated chromosome, MipZ dimers control the placement of the cell division plane by preventing FtsZ proteins from assembling into a Z-ring. MipZ proteins generate bipolar gradients that are sensitive to Par protein localization, however, it is not understood how the MipZ gradient is shaped so as to allow for the correct Z-ring placement during asymmetric cell division in C. crescentus. In this paper, we develop and analyze a mathematical model that incorporates the known interactions between Par, PopZ, and MipZ proteins and use it to test mechanisms for MipZ gradient formation. Using our model, we show that gradient-dependent ParB advection velocities in conjunction with a ParA polar recycling mechanism are sufficient to maintain a robust new pole-directed ParA dimer gradient during segregation. A "saturation of binding site" hypothesis limiting access of ParA and MipZ to the ParB complex is then necessary and sufficient to generate time-averaged bipolar MipZ protein gradients with minima that are skewed toward ParA gradient peaks at the new pole, in agreement with data. By analyzing reduced versions of the model, we show the existence of oscillatory ParA localization regimes provided that cytoplasmic PopZ oligomers interact with ParA and ParA is over-expressed. We use our model to study mechanisms by which these protein patterns may simultaneously direct proper chromosome segregation and division site placement in C. crescentus.

Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer.

Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.

Structure of the hexagonal surface layer on Caulobacter crescentus cells.

Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea1-5. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 Šstructure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 Å, and is stabilized by multiple Ca2+ ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging.