Abstract: We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster . In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

Abstract: Soft rot disease causes devastating losses to crop plants all over the world, with up to 90% loss in tropical climates. To better understand this economically important disease, we isolated four soft rot-causing Erwinia persicina strains from rotted vegetables. Notably, E. persicina has only recently been identified as a soft rot pathogen and a comprehensive genomic analysis and comparison has yet to be conducted. Here, we provide the first genomic analysis of E. persicina , compared to Pectobacterium carotovorum , P. carotovorum , and associated Erwinia plant pathogens. We found that E. persicina shares common genomic features with other Erwinia species and P. carotovorum , while having its own unique characteristics as well. The E. persicina strains examined here lack Type II and Type III secretion systems, commonly used to secrete pectolytic enzymes and evade the host immune response, respectively. E. persicina contains fewer putative pectolytic enzymes than P. carotovorum and lacks the Out cluster of the Type II secretion system while harboring a siderophore that causes a unique pink pigmentation during soft rot infections. Interestingly, a putative phenolic acid decarboxylase is present in the E. persicina strains and some soft rot pathogens, but absent in other Erwinia species, thus potentially providing an important factor for soft rot. All four E. persicina isolates obtained here and many other E. persicina genomes contain plasmids larger than 100 kbp that encode proteins likely important for adaptation to plant hosts. This research provides new insights into the possible mechanisms of soft rot disease by E. persicina and potential targets for diagnostic tools and control measures.

Abstract: Eleven Pectobacterium strains were isolated from soft rot-diseased vegetables. Here, we report their genome sequences and characteristics. Five isolates were found to be Pectobacterium versatile , while the other six were determined to be Pectobacterium brasiliense .

Abstract: The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Abstract: In conclusion, we have identified a role for the FtsEX ABC system in regulating septal PG hydrolysis. Importantly, in an accompanying report, Sham et al. ( 5 ) describe a similar regulatory role for FtsEX in cell separation in the Gram-positive pathogen Streptococcus pneumoniae , indicating this function for FtsEX is likely to be broadly conserved. Given the diversity and ubiquity of ABC systems in nature ( 1 ), the ATP-driven conformational changes in these membrane complexes likely have been adapted to regulate a variety of biological processes. Consistent with this model, sequence analysis groups FtsEX with a subclass of ABC systems comprised mainly of substrate-binding protein (SBP)-dependent importers like the maltose transporter (MalFGK 2 ) ( 1 ). The structure of MalFGK 2 , when complexed with maltose-binding protein (MalE), indicates that ATP-driven conformational changes in the transporter can alter the conformation of periplasmic MalE ( 4 ). In this case, MalE is converted from its closed, maltose-bound conformation to an open conformation that releases maltose into the outward-facing cavity of the transporter for subsequent import ( 4 ). We therefore propose that EnvC may be an SBP analog for FtsEX and envision that the conformation of EnvC is modulated similarly by the ABC system so that it interconverts between an “on” and “off” state during an ATPase cycle ( Fig. P1 ). This model is appealing for several reasons. Most significantly, it would provide a means for converting septal PG hydrolysis into a discrete process with a fixed number of PG bonds being broken per ATP molecule hydrolyzed. This process would afford the cytokinetic ring exquisite control over PG hydrolysis; such control seems highly desirable given the inherent risks involved in promoting localized PG degradation. Additionally, because FtsE interacts with FtsZ in the cytoplasm, the ATPase activity of FtsE could be coupled directly to Z-ring dynamics. Thus, the FtsEX complex could serve as a molecular governor to coordinate the rate of septal PG hydrolysis properly with the contraction of the Z-ring. Finally, in addition to connecting the Z-ring with septal PG hydrolysis, interactions of FtsX with other transmembrane components involved in the division process may help couple the activity of enzymes that synthesize PG with the cell-separation amidases. For example, the FtsEX complex may promote EnvC-activated amidase activity only when it is engaged with an active PG synthetic complex. Given the findings of Weiss and coworkers ( 2 ), we wondered if the ATP-hydrolyzing (ATPase) activity of FtsE might be required to stimulate amidase activation by EnvC. We therefore generated FtsE* variants with substitutions in the ATP-binding site and tested their ability to promote cell separation. Interestingly, when cells were grown under permissive conditions, all the FtsE* variants failed to promote normal cell separation. In addition, localization experiments showed that recruitment of EnvC and FtsX to the division site was unaffected in these cells. Furthermore, based on the results obtained by Weiss and coworkers ( 2 ) with similar mutants, we assume that the FtsE* variants described here also are normally recruited to the cytokinetic ring. Thus, FtsE*X–EnvC complexes likely form at the division site in cells expressing the ftsE * alleles but fail to function or function poorly in the septal PG-splitting process. We therefore infer that the ATPase activity of the FtsEX complex plays an important role in promoting amidase activation by EnvC at the septum. An attractive possibility is that ATP hydrolysis by FtsE is used to induce conformational changes in FtsX similar to those observed in other ABC systems ( 1 ) to regulate EnvC activity in the periplasm allosterically ( Fig. P1 ). Weiss and coworkers ( 2 ) recently showed that FtsE residues in protein motifs predicted to be important for ATP hydrolysis are required for FtsEX to function in cell division. Interestingly, however, these residues were not found to be needed for the stability of FtsE or the recruitment of either FtsE or FtsX to the division site ( 2 ). In addition, unlike ftsEX -null mutants, division proteins downstream of FtsEX in the localization hierarchy were recruited to the cytokinetic ring in the presence of these predicted ATPase-defective variants ( 2 ). Thus, by all indications, complete cytokinetic rings formed in the cells producing the FtsE variants, but cell constriction could not occur in the absence of ATP hydrolysis by FtsEX ( 2 ). We thought of several likely reasons for the FtsEX requirement. FtsEX may be required for ( i ) EnvC stability, ( ii ) the export of EnvC to the periplasm, or ( iii ) the recruitment of EnvC to the division site. Two techniques, cell fractionation and immunoblot analysis, indicated that EnvC accumulates stably in the periplasm of cells lacking FtsEX, demonstrating that FtsEX is not required for EnvC stability or its export. However, subcellular localization experiments revealed that EnvC failed to be recruited to the division site when cells were depleted of FtsEX. This result indicated that FtsEX is needed for EnvC localization to the division site and suggested that the two proteins might interact. Indeed, further experiments demonstrated that EnvC and FtsX interact directly and that the interaction occurs between the N-terminal coiled-coil domain of EnvC and a large periplasmic loop domain of FtsX. This interaction was shown to be important for EnvC localization in bacterial cells using FtsX variants with deletions of various portions of the loop domain. All the FtsX deletion variants, including one lacking almost the entire loop, remained capable of localizing to the division site, indicating that the loop domain is not important for the localization of FtsX itself. However, even the FtsX variant with the smallest loop deletion tested failed to recruit EnvC to the cytokinetic ring. Using a genetic screen designed to identify factors involved in cell-wall assembly, we found that mutations in both ftsEX and envC are lethal when combined with a loss-of-function mutation in a gene coding for a cell-wall synthesis enzyme. These results suggested that FtsEX and EnvC might participate in the same biochemical pathway. Database searches also hinted at this possibility, because a few organisms appear to encode ftsE , ftsX , and envC in the same unit of linked genes (operon), although E. coli does not. We found that cells lacking FtsEX (FtsEX − cells), when grown under permissive conditions, show the same cell-separation defect displayed by EnvC − cells. This result further solidified the connection between FtsEX and EnvC. Thus, the genetic data pointed toward FtsEX being necessary for EnvC to activate the amidases at the division site. Most bacteria surround themselves with a polysaccharide (cell wall) matrix called peptidoglycan (PG) that is essential for cellular integrity ( Fig. P1 ). During cytokinesis in many bacteria, the FtsZ protein forms a ring structure (the Z-ring), which organizes the synthesis of new PG material that eventually will fortify the daughter cell poles. This material is thought to be shared initially by the developing daughter cells and must be split to facilitate outer membrane constriction and daughter cell separation ( Fig. P1 ). Cell-wall splitting is mediated by AmiA, AmiB, and AmiC, cell-wall hydrolase enzymes exported to the space between the inner and outer cell membranes (periplasm). These enzymes have PG amidase activity that hydrolyzes amide bonds in the PG structure to destroy the crosslinks that hold the meshwork together. This activity must be controlled tightly to prevent these factors from creating breaches in the cell wall that can lead to cell lysis. Part of this regulation appears to rely on the weak intrinsic activity of the amidases in the absence of factors that promote their hydrolytic function. To hydrolyze PG efficiently, they require activation by EnvC and NlpD, proteins with LytM domains associated with the cytokinetic ring ( 3 ). EnvC specifically activates AmiA and AmiB, and NlpD specifically activates AmiC. ATP-binding cassette (ABC) transporters are membrane protein complexes found in all living organisms and use ATP molecules to power the movement of substrate molecules across membranes ( 1 ). To perform this function, these transporters use ATP-induced conformational changes in their nucleotide-binding domains (NBDs) (i.e., the amino acid sequences that bind to ATP) to alter the conformation of their transmembrane domain (TMD) components ( 1 ). This change causes the transport cavity formed by the TMDs to cycle between inward-facing and outward-facing conformations, thus driving transport by an “alternating access” mechanism ( 1 ). In a wide variety of bacteria, the ABC transporter-like FtsEX complex is an important component of the cytokinetic ring structure that drives cell division ( 2 ). FtsE is the NBD component of the complex, and FtsX is the TMD subunit. The role of FtsEX in cell division has remained a mystery for many years. Here, we report that the FtsEX complex is required for the activation of cell-wall hydrolysis (i.e., cell-wall breakdown) at the division site of the bacterium Escherichia coli to promote the separation of new daughter cells. Our results suggest the attractive possibility that FtsEX accomplishes this function using ATP hydrolysis to directly control the conformation of EnvC, a critical activator of cell-wall hydrolysis associated with the FtsEX complex on the outer surface of the cell membrane ( Fig. P1 ).

Abstract: The ejection of material from Mars is thought to be caused by large impacts that would heat much of the ejecta to high temperatures. Images of the magnetic field of martian meteorite ALH84001 reveal a spatially heterogeneous pattern of magnetization associated with fractures and rock fragments. Heating the meteorite to 40°C reduces the intensity of some magnetic features, indicating that the interior of the rock has not been above this temperature since before its ejection from the surface of Mars. Because this temperature cannot sterilize most bacteria or eukarya, these data support the hypothesis that meteorites could transfer life between planets in the solar system.

Abstract: Cell division in Escherichia coli begins with the polymerization of FtsZ into a ring‐like structure, the Z‐ring, at midcell. All other division proteins are thought to require the Z‐ring for recruitment to the future division site. Here, it is reported that the Z‐ring associated proteins ZapA and ZapB form FtsZ‐independent structures at midcell. Upon Z‐ring disruption by the FtsZ polymerization antagonist SulA, ZapA remained at midcell as a cloud‐like accumulation. Using ZapA(N60Y), a variant defective for interaction with FtsZ, it was established that these ZapA structures form without a connection to the Z‐ring. Furthermore, midcell accumulations of GFP‐ZapA(N60Y) often preceded Z‐rings at midcell and required ZapB to assemble, suggesting that ZapB polymers form the foundation of these structures. In the absence of MatP, a DNA‐binding protein that links ZapB to the chromosomal terminus region, cloud‐like ZapA structures still formed but failed to track with the chromosome terminus and did not consistently precede FtsZ at midcell. Taken together, the results suggest that FtsZ‐independent structures of ZapA–ZapB provide additional positional cues for Z‐ring formation and may help coordinate its assembly with chromosome replication and segregation.