An Aerobic Exercise: Defining the Roles of Pseudomonas aeruginosa Terminal Oxidases

The opportunistic pathogen Pseudomonas aeruginosa encodes a large and diverse complement of aerobic terminal oxidases, which is thought to contribute to its ability to thrive in settings with low oxygen availability. In this issue, Arai et al. (J. Bacteriol. 196:4206–4215, 2014, doi:http://dx.doi.org/10.1128/JB.02176-14) present a thorough characterization of these five complexes, enabling a more detailed understanding of aerobic respiration in this organism.     

The Salmonella Type III Secretion System Virulence Effector Forms a New Hexameric Chaperone Assembly for Export of Effector/Chaperone Complexes

Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane''s cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.     

Bacteria in Solitary Confinement

Even in clonal bacterial cultures, individual bacteria can show substantial stochastic variation, leading to pitfalls in the interpretation of data derived from millions of cells in a culture. In this issue of the Journal of Bacteriology, as part of their study on osmoadaptation in a cyanobacterium, Nanatani et al. describe employing an ingenious microfluidic device that gently cages individual cells (J Bacteriol 197:676–687, 2015, http://dx.doi.org/10.1128/JB.02276-14). The device is a welcome addition to the toolkit available to probe the responses of individual cells to environmental cues.     

Death by Cystine: an Adverse Emergent Property from a Beneficial Series of Reactions

In this issue of the Journal of Bacteriology, Chonoles Imlay et al. (K. R. Chonoles Imlay, S. Korshunov, and J. A. Imlay, J Bacteriol 197:3629–3644, 2015, http://dx.doi.org/10.1128/JB.00277-15) show that oxidative stress kills sulfur-restricted Escherichia coli grown with sublethal H₂O₂ when challenged with cystine. Killing requires rapid and seemingly unregulated cystine transport and equally rapid cystine reduction to cysteine. Cysteine export completes an energy-depleting futile cycle. Each reaction of the cycle could be beneficial. Together, a cystine-mediated vulnerability emerges during the transition from a sulfur-restricted to a sulfur-replete environment, perhaps because of complexities of sulfur metabolism.     

Fifty Ways To Inhibit Motility via Cyclic Di-GMP: the Emerging Pseudomonas aeruginosa Swarming Story

There are numerous ways by which cyclic dimeric GMP (c-di-GMP) inhibits motility. Kuchma et al. (S. L. Kuchma, N. J. Delalez, L. M. Filkins, E. A. Snavely, J. P. Armitage, and G. A. O''Toole, J. Bacteriol. 197:420–430, 2015, http://dx.doi.org/10.1128/JB.02130-14) offer a new, previously unseen way of swarming motility inhibition in Pseudomonas aeruginosa PA14. This bacterium possesses a single flagellum with one rotor and two sets of stators, only one of which can provide torque for swarming. The researchers discovered that elevated levels of c-di-GMP inhibit swarming by skewing stator selection in favor of the nonfunctional, “bad” stators.     

Another Look at Mutations in Ribosomal Protein S4 Lends Strong Support to the Domain Closure Model

Ribosomes employ a “kinetic discrimination” mechanism, in which correct substrates are incorporated more rapidly than incorrect ones. The structural basis of this mechanism may involve 30S domain closure, a global conformational change that coincides with codon recognition. In a direct screen for fidelity-altering mutations, Agarwal and coworkers (D. Agarwal, D. Kamath, S. T. Gregory, and M. O''Connor, J Bacteriol 197:1017–1025, 2015, doi:10.1128/JB.02485-14) isolated mutations that progressively truncate the C terminus of S4. All of these promote miscoding and undoubtedly destabilize the S4-S5 interface, consistent with the domain closure model.     

Reinterpreting Long-Term Evolution Experiments: Is Delayed Adaptation an Example of Historical Contingency or a Consequence of Intermittent Selection?

Van Hofwegen et al. demonstrated that Escherichia coli rapidly evolves the ability to use citrate when long selective periods are provided (D. J. Van Hofwegen, C. J. Hovde, and S. A. Minnich, J Bacteriol 198:1022–1034, 2016, http://dx.doi.org/10.1128/JB.00831-15). This contrasts with the extreme delay (15 years of daily transfers) seen in the long-term evolution experiments of Lenski and coworkers. Their idea of “historical contingency” may require reinterpretation. Rapid evolution seems to involve selection for duplications of the whole cit locus that are too unstable to contribute when selection is provided in short pulses.     

Rethinking the Hierarchy of Sugar Utilization in Bacteria

Bacteria are known to consume some sugars over others, although recent work reported by Koirala and colleagues in this issue of the Journal of Bacteriology (S. Koirala, X. Wang, and C. V. Rao, J Bacteriol 198:386–393, 2016, http://dx.doi.org/10.1128/JB.00709-15) revealed that individual cells do not necessarily follow this hierarchy. By studying the preferential consumption of l-arabinose over d-xylose in Escherichia coli, those authors found that subpopulations consume one, the other, or both sugars through cross-repression between utilization pathways. Their findings challenge classic assertions about established hierarchies and can guide efforts to engineer the simultaneous utilization of multiple sugars.     

Ca2+ block and flickering both contribute to the negative slope of the IV curve in BK channels

Single-channel current–voltage (IV) curves of human large-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channels are quite linear in 150 mM KCl. In the presence of Ca²⁺ and/or Mg²⁺, they show a negative slope conductance at high positive potentials. This is generally explained by a Ca²⁺/Mg²⁺ block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca²⁺ and 2.5 mM Mg²⁺ per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca²⁺/Mg²⁺. In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca²⁺/Mg²⁺. The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.     

Controlling the Connections of Cells to the Biofilm Matrix

The importance of cyclic di-GMP (c-di-GMP) and its control of biofilm matrix assembly and production has been a focal point of researchers in recent history. In this issue, Cooley et al. (Cooley RB, Smith TJ, Leung W, Tierney V, Borlee BR, O’Toole GA, Sondermann H, J Bacteriol 198:66–77, http://dx.doi.org/10.1128/JB.00369-15) demonstrate that two c-di-GMP controlled features of Pseudomonas aeruginosa, the periplasmic protease LapG and the surface adhesin CdrA, are linked. CdrA is shown to be a substrate of LapG, with LapG activity controlled by intracellular c-di-GMP levels. This commentary discusses the significance of this finding.     

Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers

Large-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channels display near linear current–voltage (I-V) plots for voltages between −100 and +100 mV, with an increasing sublinearity for more positive potentials. As is the case for many types of channels, BK channels are blocked at positive potentials by intracellular Ca²⁺ and Mg²⁺. This fast block progressively reduces single-channel conductance with increasing voltage, giving rise to a negative slope in the I-V plots beyond about +120 mV, depending on the concentration of the blockers. In contrast to these observations of pronounced differences in the magnitudes and shapes of I-V plots in the absence and presence of intracellular blockers, Schroeder and Hansen (2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) have reported identical I-V plots in the absence and presence of blockers for BK channels, with both plots having reduced conductance and negative slopes, as expected for blockers. Schroeder and Hansen included both Ca²⁺ and Mg²⁺ in the intracellular solution rather than a single blocker, and they also studied BK channels expressed from α plus β1 subunits, whereas most previous studies used only α subunits. Although it seems unlikely that these experimental differences would account for the differences in findings between previous studies and those of Schroeder and Hansen, we repeated the experiments using BK channels comprised of α plus β1 subunits with joint application of 2.5 mM Ca²⁺ plus 2.5 mM Mg²⁺, as Schroeder and Hansen did. In contrast to the findings of Schroeder and Hansen of identical I-V plots, we found marked differences in the single-channel I-V plots in the absence and presence of blockers. Consistent with previous studies, we found near linear I-V plots in the absence of blockers and greatly reduced currents and negative slopes in the presence of blockers. Hence, studies of conductance mechanisms for BK channels should exclude intracellular Ca²⁺/Mg²⁺, as they can reduce conductance and induce negative slopes.     

Plakophilin-2 Promotes Activation of Epidermal Growth Factor Receptor

While growth factor-driven dimerization of receptor tyrosine kinases (RTKs) is a simple and intuitive mechanism of activating RTKs, K.-I. Arimoto et al. (Mol. Cell. Biol. 34:3843–3854, 2014, doi:10.1128/MCB.00758-14) describe a novel means of promoting the activity of RTKs. Namely, plakophilin-2 (PKP2) associates with the epidermal growth factor receptor (EGFR) and enhances its ligand-dependent and ligand-independent activity. This discovery suggests that antagonizing PKP2 may be a new therapeutic opportunity to combat tumors in which activation of EGFR contributes to pathogenesis.     

Characterization and Localization of the Campylobacter jejuni Transformation System Proteins CtsE,CtsP, and CtsX

The human pathogen Campylobacter jejuni is naturally competent for transformation with its own DNA. Genes required for efficient transformation in C. jejuni include those similar to components of type II secretion systems found in many Gram-negative bacteria (R. S. Wiesner, D. R. Hendrixson, and V. J. DiRita, J Bacteriol 185:5408–5418, 2003, http://dx.doi.org/10.1128/JB.185.18.5408-5418.2003). Two of these, ctsE and ctsP, encode proteins annotated as putative nucleotide binding nucleoside triphosphatases (NTPases) or nucleoside triphosphate (NTP) binding proteins. Here we demonstrate that the nucleotide binding motifs of both proteins are essential for their function in transformation of C. jejuni. Localization experiments demonstrated that CtsE is a soluble protein while CtsP is membrane associated in C. jejuni. A bacterial two-hybrid screen identified an interaction between CtsP and CtsX, an integral membrane protein also required for transformation. Topological analysis of CtsX by the use of LacZ and PhoA fusions demonstrated it to be a bitopic, integral membrane protein with a cytoplasmic amino terminus and a periplasmic carboxyl terminus. Notwithstanding its interaction with membrane-localized CtsX, CtsP inherently associates with the membrane, requiring neither CtsX nor several other Cts proteins for this association.     

Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact of C. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863–3875, 2013, http://dx.doi.org/10.1128/JB.00369-13; J. Phetcharaburanin et al., Mol Microbiol 92:1025–1038, 2014, http://dx.doi.org/10.1111/mmi.12611), there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore''s outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface of C. difficile spores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets.     

Complete Genome Sequence Analysis of Two Pseudomonas plecoglossicida Phages,Potential Therapeutic Agents

Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416–1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33–39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+ and Zn2+) and water coordination on the structure and properties of l-histidine and zwitterionic l-histidine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of l-histidine is examined. The effect of metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺) and water on structures of His·M(H₂O)m, m = 0.1 complexes have been determined theoretically employing density functional theories using extended basis sets. Of the five stable complexes investigated the relative stability of the gas-phase complexes computed with DFT methods (with one exception of K⁺ systems) suggest metallic complexes of the neutral l-histidine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of l-histidine in the presence of the metal cations Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to l-histidine is exhibited by the Cu²⁺ cation. The computed Gibbs energies ΔG are negative, span a rather broad energy interval (from ?130 to ?1,300 kJ/mol), and upon hydration are appreciably lowered.     

Population Bottlenecks and Pathogen Extinction: “Make This Everyone's Mission to Mars,Including Yours”

Kapusinszky et al. (J Virol 89:8152–8161, 2015, http://dx.doi.org/10.1128/JVI.00671-15) report that host population bottlenecks may result in pathogen extinction, which provides a compelling argument for an alternative approach to vaccination for the control of virus spread. By comparing the prevalence levels of three viral pathogens in two populations of African green monkeys (AGMs) (Chlorocebus sabaeus) from Africa and two Caribbean Islands, they convincingly show that a major host bottleneck resulted in the eradication of select pathogens from a given host.