The divergent early divisome: is there a functional core?

The bacterial divisome is a complex nanomachine that drives cell division and separation. The essentiality of these processes leads to the assumption that proteins with core roles will be strictly conserved across all bacterial genomes. However, recent studies in diverse proteobacteria have revealed considerable variation in the early divisome compared with Escherichia coli. While some proteins are highly conserved, their specific functions and interacting partners vary. Meanwhile, different subphyla use clade-specific proteins with analogous functions. Thus, instead of focusing on gene conservation, we must also explore how key functions are maintained during early division by diverging protein networks. An enhanced awareness of these complex genetic networks will clarify the physical and evolutionary constraints of bacterial division.

A peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens.

During growth, bacteria remodel and recycle their peptidoglycan (PG). A key family of PG-degrading enzymes is the lytic transglycosylases, which produce anhydromuropeptides, a modification that caps the PG chains and contributes to bacterial virulence. Previously, it was reported that the polar-growing Gram-negative plant pathogen Agrobacterium tumefaciens lacks anhydromuropeptides. Here, we report the identification of an enzyme, MdaA (MurNAc deacetylase A), which specifically removes the acetyl group from anhydromuropeptide chain termini in A. tumefaciens, resolving this apparent anomaly. A. tumefaciens lacking MdaA accumulates canonical anhydromuropeptides, whereas MdaA was able to deacetylate anhydro-N-acetyl muramic acid in purified sacculi that lack this modification. As for other PG deacetylases, MdaA belongs to the CE4 family of carbohydrate esterases but harbors an unusual Cys residue in its active site. MdaA is conserved in other polar-growing bacteria, suggesting a possible link between PG chain terminus deacetylation and polar growth.

Agrobacterium tumefaciens: a Transformative Agent for Fundamental Insights into Host-Microbe Interactions, Genome Biology, Chemical Signaling, and Cell Biology.

Agrobacterium tumefaciens incites the formation of readily visible macroscopic structures known as crown galls on plant tissues that it infects. Records from biologists as early as the 17th century noted these unusual plant growths and began examining the basis for their formation. These studies eventually led to isolation of the infectious agent, A. tumefaciens, and decades of study revealed the remarkable mechanisms by which A. tumefaciens causes crown gall through stable horizontal genetic transfer to plants. This fundamental discovery generated a barrage of applications in the genetic manipulation of plants that is still under way. As a consequence of the intense study of A. tumefaciens and its role in plant disease, this pathogen was developed as a model for the study of critical processes that are shared by many bacteria, including host perception during pathogenesis, DNA transfer and toxin secretion, bacterial cell-cell communication, plasmid biology, and more recently, asymmetric cell biology and composite genome coordination and evolution. As such, studies of A. tumefaciens have had an outsized impact on diverse areas within microbiology and plant biology that extend far beyond its remarkable agricultural applications. In this review, we attempt to highlight the colorful history of A. tumefaciens as a study system, as well as current areas that are actively demonstrating its value and utility as a model microorganism.

Activation of ChvG-ChvI regulon by cell wall stress confers resistance to ß-lactam antibiotics and initiates surface spreading in Agrobacterium tumefaciens.

A core component of nearly all bacteria, the cell wall is an ideal target for broad spectrum antibiotics. Many bacteria have evolved strategies to sense and respond to antibiotics targeting cell wall synthesis, especially in the soil where antibiotic-producing bacteria compete with one another. Here we show that cell wall stress caused by both chemical and genetic inhibition of the essential, bifunctional penicillin-binding protein PBP1a prevents microcolony formation and activates the canonical host-invasion two-component system ChvG-ChvI in Agrobacterium tumefaciens. Using RNA-seq, we show that depletion of PBP1a for 6 hours results in a downregulation in transcription of flagellum-dependent motility genes and an upregulation in transcription of type VI secretion and succinoglycan biosynthesis genes, a hallmark of the ChvG-ChvI regulon. Depletion of PBP1a for 16 hours, results in differential expression of many additional genes and may promote a stress response, resembling those of sigma factors in other bacteria. Remarkably, the overproduction of succinoglycan causes cell spreading and deletion of the succinoglycan biosynthesis gene exoA restores microcolony formation. Treatment with cefsulodin phenocopies depletion of PBP1a and we correspondingly find that chvG and chvI mutants are hypersensitive to cefsulodin. This hypersensitivity only occurs in response to treatment with ß-lactam antibiotics, suggesting that the ChvG-ChvI pathway may play a key role in resistance to antibiotics targeting cell wall synthesis. Finally, we provide evidence that ChvG-ChvI likely has a conserved role in conferring resistance to cell wall stress within the Alphaproteobacteria that is independent of the ChvG-ChvI repressor ExoR.

Induction of AmpC-Mediated ß-Lactam Resistance Requires a Single Lytic Transglycosylase in Agrobacterium tumefaciens.

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here, we use a reverse-genetics approach to identify genes involved in ampicillin resistance, with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased ß-lactamase production, a response dependent on the broad-spectrum ß-lactamase AmpC and its transcription factor, AmpR. Loss of the putative ampD orthologue atu2113 led to constitutive production of AmpC-dependent ß-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent ß-lactamase activity, and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 ΔmltB3 transforms plants with efficiency comparable to that of the wild type but can be cleared with sublethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium. IMPORTANCE Agrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in ß-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit ß-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

Unipolar Peptidoglycan Synthesis in the Rhizobiales Requires an Essential Class A Penicillin-Binding Protein.

Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens. The development of fluorescent d-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation. IMPORTANCE While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales. Our findings suggest that penicillin-binding protein 1a, along with a subset of ld-transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales.

Diversification of LytM Protein Functions in Polar Elongation and Cell Division of Agrobacterium tumefaciens.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

d-canavanine affects peptidoglycan structure, morphogenesis and fitness in Rhizobiales.

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here, we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyses production of dl-CAN from l-CAN, which is produced by many legumes. Importantly, d-CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that d-CAN alters dramatically the PG structure of Rhizobiales (e.g., Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens, we demonstrated that the detrimental effect of d-CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness.

Isolation and Characterization T4- and T7-Like Phages that Infect the Bacterial Plant Pathogen Agrobacterium tumefaciens.

In the rhizosphere, bacteria-phage interactions are likely to have important impacts on the ecology of microbial communities and microbe-plant interactions. To better understand the dynamics of Agrobacteria-phage interactions, we have isolated diverse bacteriophages which infect the bacterial plant pathogen, Agrobacterium tumefaciens. Here, we complete the genomic characterization of Agrobacteriumtumefaciens phages Atu_ph04 and Atu_ph08. Atu_ph04-a T4-like phage belonging to the Myoviridae family-was isolated from waste water and has a 143,349 bp genome that encodes 223 predicted open reading frames (ORFs). Based on phylogenetic analysis and whole-genome alignments, Atu_ph04 is a member of a newly described T4 superfamily that contains other Rhizobiales-infecting phages. Atu_ph08, a member of the Podoviridae T7-like family, was isolated from waste water, has a 59,034 bp genome, and encodes 75 ORFs. Based on phylogenetic analysis and whole-genome alignments, Atu_ph08 may form a new T7 superfamily which includes Sinorhizobium phage PCB5 and Ochrobactrum phage POI1126. Atu_ph08 is predicted to have lysogenic activity, as we found evidence of an integrase and several transcriptional repressors with similarity to proteins in transducing phage P22. Together, this data suggests that Agrobacterium phages are diverse in morphology, genomic content, and lifestyle.

Higher functionality of bacterial plasmid DNA in water after peracetic acid disinfection compared with chlorination.

Peracetic acid (PAA) is an emerging disinfectant with a low disinfection by-product formation potential, but how PAA destroys gene function after killing bacteria remains to be studied. Bacterial plasmid DNA is a mobile genetic element that often harbors undesirable genes encoding antibiotic resistance and virulence factors. Even though PAA efficiently kills bacteria, bacterial plasmids and other mobile genetic elements might still be intact and functional after PAA disinfection, posing potential public health and environmental risks. This study evaluated the impact of PAA disinfection on the functionality of plasmid DNA in vivo and compared the results with those from chlorination. We delivered a plasmid DNA harboring two antibiotic resistance genes to Escherichia coli TOP10 to form an antibiotic-resistant bacterium (ARB). The planktonic ARB was treated with PAA and chlorine to find the minimum doses inhibiting the regrowth of the strain. PAA and chlorine stopped the regrowth at 8 ±â€¯1 mg PAA·L-1 and 20 ±â€¯9 mg Cl2·L-1, respectively. The functionality of the plasmid DNA after PAA and chlorine disinfection was then determined at higher doses in vivo. Neither PAA nor chlorine completely destroyed the plasmid DNA. However, chlorine was more efficient than PAA in eliminating the plasmid DNA. PAA at 25 mg PAA·L-1 reduced the transforming activity of the plasmid DNA by less than 0.3 log10 units, whereas chlorine at 25 mg Cl2·L-1 reduced the transforming activity by approximately 1.7 log10 units. Chlorine had a more pronounced impact on the functionality of the plasmid DNA because it oxidizes or destroys bacterial components including plasmid DNA faster than PAA. In addition, environmental scanning electron microscopy shows that chlorination desiccated the cells resulting in the flat cellular structure and possibly more complete loss of plasmid DNA, whereas PAA disinfection had a less impact on cell structure and morphology. This study demonstrates that more plasmid DNA remains functional in water after PAA disinfection than after chlorination. These functional genetic elements could be acquired by other microorganisms via horizontal gene transfer to pose potential public health and environmental risks.

An Essential Regulator of Bacterial Division Links FtsZ to Cell Wall Synthase Activation.

Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function; however, it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here, we show that FzlA, an FtsZ-binding protein and essential regulator of constriction in Caulobacter crescentus, helps link FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell-wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another α-proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA helps link FtsZ to cell wall remodeling and is required for signaling to both activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division.

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re-direct peptidoglycan biosynthesis to mid-cell during cell division in polar-growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid-cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid-cell, exhibit GTPase activity and form co-polymers, only one, FtsZAT , is required for cell division. We find that FtsZAT is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid-cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZAT in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid-cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.

Inhibition of regrowth of planktonic and biofilm bacteria after peracetic acid disinfection.

Peracetic acid (PAA) is a promising alternative to chlorine for disinfection; however, bacterial regrowth after PAA disinfection is poorly understood. This study compared the regrowth of bacteria (Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bacillus sp.) after disinfection with PAA or free chlorine. In the absence of organic matter, PAA and free chlorine prevented the regrowth of planktonic cells of P. aeruginosa PAO1 at C·t (= disinfectant concentration × contact time) doses of (28.5 ±â€¯9.8) mg PAA·min·L-1 and (22.5 ±â€¯10.6) mg Cl2·min·L-1, respectively, suggesting that they had comparable efficiencies in preventing the regrowth of planktonic bacteria. For comparison, the minimum C·t doses of PAA and free chlorine to prevent the regrowth of P. aeruginosa PAO1 biofilm cells in the absence of organic matter were (14,000 ±â€¯1,732) mg PAA·min·L-1 and (6,500 ±â€¯2,291) mg Cl2·min·L-1, respectively. PAA was less effective than free chlorine in killing bacteria within biofilms in the absence of organic matter most likely because PAA reacts with biofilm matrix constituents slower than free chlorine. In the presence of organic matter, although the bactericidal efficiencies of both disinfectants significantly decreased, PAA was less affected due to its slower reaction with organic matter and/or slower self-decomposition. For instance, in a dilute Lysogeny broth-Miller, the minimum concentrations of PAA and free chlorine to prevent the regrowth of planktonic P. aeruginosa PAO1 were 20 mg PAA·L-1 and 300 mg Cl2·L-1, respectively. While both disinfectants are strong oxidants disrupting cell membrane, environmental scanning electron microscopy (ESEM) revealed that PAA made holes in the center of the cells, whereas free chlorine desiccated the cells. Overall, this study shows that PAA is a powerful disinfectant to prevent bacterial regrowth even in the presence of organic matter.

Larger Than Life: Isolation and Genomic Characterization of a Jumbo Phage That Infects the Bacterial Plant Pathogen, Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease, leading to the damage of agriculturally-important crops. As part of an effort to discover new phages that can potentially be used as biocontrol agents to prevent crown gall disease, we isolated and characterized phage Atu_ph07 from Sawyer Creek in Springfield, MO, using the virulent Agrobacterium tumefaciens strain C58 as a host. After surveying its host range, we found that Atu_ph07 exclusively infects Agrobacterium tumefaciens. Time-lapse microscopy of A. tumefaciens cells subjected to infection at a multiplicity of infection (MOI) of 10 with Atu_ph07 reveals that lysis occurs within 3 h. Transmission electron microscopy (TEM) of virions shows that Atu_ph07 has a typical Myoviridae morphology with an icosahedral head, long tail, and tail fibers. The sequenced genome of Atu_ph07 is 490 kbp, defining it as a jumbo phage. The Atu_ph07 genome contains 714 open reading frames (ORFs), including 390 ORFs with no discernable homologs in other lineages (ORFans), 214 predicted conserved hypothetical proteins with no assigned function, and 110 predicted proteins with a functional annotation based on similarity to conserved proteins. The proteins with predicted functional annotations share sequence similarity with proteins from bacteriophages and bacteria. The functionally annotated genes are predicted to encode DNA replication proteins, structural proteins, lysis proteins, proteins involved in nucleotide metabolism, and tRNAs. Characterization of the gene products reveals that Atu_ph07 encodes homologs of 16 T4 core proteins and is closely related to Rak2-like phages. Using ESI-MS/MS, the majority of predicted structural proteins could be experimentally confirmed and 112 additional virion-associated proteins were identified. The genomic characterization of Atu_ph07 suggests that this phage is lytic and the dynamics of Atu_ph07 interaction with its host indicate that this phage may be suitable for inclusion in a phage cocktail to be used as a biocontrol agent.

An aryl-homoserine lactone quorum-sensing signal produced by a dimorphic prosthecate bacterium.

Many species of Proteobacteria produce acyl-homoserine lactone (AHL) compounds as quorum-sensing (QS) signals for cell density-dependent gene regulation. Most known AHL synthases, LuxI-type enzymes, produce fatty AHLs, and the fatty acid moiety is derived from an acyl-acyl carrier protein (ACP) intermediate in fatty acid biosynthesis. Recently, a class of LuxI homologs has been shown to use CoA-linked aromatic or amino acid substrates for AHL synthesis. By using an informatics approach, we found the CoA class of LuxI homologs exists primarily in α-Proteobacteria. The genome of Prosthecomicrobium hirschii, a dimorphic prosthecate bacterium, possesses a luxI-like AHL synthase gene that we predicted to encode a CoA-utilizing enzyme. We show the P. hirschii LuxI homolog catalyzes synthesis of phenylacetyl-homoserine lactone (PA-HSL). Our experiments show P. hirschii obtains phenylacetate from its environment and uses a CoA ligase to produce the phenylacetyl-CoA substrate for the LuxI homolog. By using an AHL degrading enzyme, we showed that PA-HSL controls aggregation, biofilm formation, and pigment production in P. hirschii These findings advance a limited understanding of the CoA-dependent AHL synthases. We describe how to identify putative members of the class, we describe a signal synthesized by using an environmental aromatic acid, and we identify phenotypes controlled by the aryl-HSL.

Cell Wall Biogenesis During Elongation and Division in the Plant Pathogen Agrobacterium tumefaciens.

A great diversity of bacterial cell shapes can be found in nature, suggesting that cell wall biogenesis is regulated both spatially and temporally. Although Agrobacterium tumefaciens has a rod-shaped morphology, the mechanisms underlying cell growth are strikingly different than other well-studied rod-shaped bacteria including Escherichia coli. Technological advances, such as the ability to deplete essential genes and the development of fluorescent D-amino acids, have enabled recent advances in our understanding of cell wall biogenesis during cell elongation and division of A. tumefaciens. In this review, we address how the field has evolved over the years by providing a historical overview of cell elongation and division in rod-shaped bacteria. Next, we summarize the current understanding of cell growth and cell division processes in A. tumefaciens. Finally, we highlight the need for further research to answer key questions related to the regulation of cell wall biogenesis in A. tumefaciens.

A novel whole-cell biosensor of Pseudomonas aeruginosa to monitor the expression of quorum sensing genes.

A novel whole-cell biosensor was developed to noninvasively and simultaneously monitor the in situ genetic activities of the four quorum sensing (QS) networks in Pseudomonas aeruginosa PAO1, including the las, rhl, pqs, and iqs systems. P. aeruginosa PAO1 is a model bacterium for studies of biofilm and pathogenesis while both processes are closely controlled by the QS systems. This biosensor worked well by selectively monitoring the expression of one representative gene from each network. In the biosensor, the promoter regions of lasI, rhlI, pqsA, and ambB (QS genes) controlled the fluorescent reporter genes of Turbo YFP, mTag BFP2, mNEON Green, and E2-Orange, respectively. The biosensor was successful in monitoring the impact of an important environmental factor, salt stress, on the genetic regulation of QS networks. High salt concentrations (≥ 20 g·L-1) significantly downregulated rhlI, pqsA, and ambB after the biosensor was incubated for 17 h to 18 h at 37 °C, resulting in slow bacterial growth.

Absence of the Min System Does Not Cause Major Cell Division Defects in Agrobacterium tumefaciens.

In A. tumefaciens, the essential FtsZ protein is located at the growth pole before shifting to the mid-cell right before division. Loss of FtsZ causes a halt in cell separation and lysis of cells. To understand how FtsZ polymerization is regulated to properly localize the FtsZ ring at the mid-cell, we have conducted a systematic characterization of the Min system in A. tumefaciens. Our findings indicate that the Min system is not required for cell survival. Yet, we find that the deletion of either minE or minCDE results in a broad cell size distribution, including an increase in the proportion of short and long cells. We observe that the site of constriction is misplaced in the minE or minCDE deletion strains allowing for short cells to arise from sites of constriction near the cell poles. Remarkably, the short cells are viable and contain DNA. In order to observe chromosome replication and segregation in these strains, YFP-ParB is used as a proxy to track the origin of replication as cells elongate and divide. In the absence of the Min proteins, duplication and segregation of the origin of replication is frequently delayed. Taken together, our data suggest that the Min system contributes to the proper regulation of FtsZ placement and subsequent cell division. Furthermore, the failure to precisely place FtsZ rings at mid-cell in the min mutants impacts other cell cycle features including chromosome segregation.

Thermodynamic properties of an emerging chemical disinfectant, peracetic acid.

Peracetic acid (PAA or CH3COOOH) is an emerging disinfectant with a low potential to form carcinogenic disinfection by-products (DBPs). Basic thermodynamic properties of PAA are, however, absent or inconsistently reported in the literature. This review aimed to summarize important thermodynamic properties of PAA, including standard Gibbs energy of formation and oxidation-reduction (redox) potential. The standard Gibbs energies of formation of CH3COOOH(aq), CH3COOOH(g), CH3COOOH(l), and CH3COOO(aq)- are -299.41kJ·mol-1, -283.02kJ·mol-1, -276.10kJ·mol-1, and -252.60kJ·mol-1, respectively. The standard redox potentials of PAA are 1.748V and 1.005V vs. standard hydrogen electrode (SHE) at pH 0 and pH 14, respectively. Under biochemical standard state conditions (pH 7, 25°C, 101,325Pa), PAA has a redox potential of 1.385V vs. SHE, higher than many disinfectants. Finally, the environmental implications of the thermodynamic properties of PAA were systematically discussed. Those properties can be used to predict the physicochemical and biological behavior of aquatic systems exposed to PAA.

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.