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Winning the war against resistant bacteria will require a change of paradigm in antibiotic discovery. A promising new direction is the targeting of non-essential pathways required for successful infection, such as quorum-sensing, virulence, and biofilm formation. Similarly important will be strategies to prevent or revert antibiotic resistance. Here, we argue that the (p)ppGpp signaling pathway should be a prime target of this effort, since its inactivation could potentially achieve all these goals simultaneously. The hyperphosphorylated guanine nucleotide (p)ppGpp is an ancient and universal second messenger of bacteria that has pleotropic effects on the physiology of these organisms. Initially described as a stress signal-an alarmone-it is now clear that (p)ppGpp plays a more general and fundamental role in bacterial adaptation, by integrating multiple internal and environmental signals to establish the optimal balance between growth and maintenance functions at any given time. Given such centrality, perturbation of the (p)ppGpp pathway will affect bacteria in multiple ways, from the ability to adjust metabolism to the available nutrients to the capacity to differentiate into developmental forms adapted to colonize different niches. Here, we provide an overview of the (p)ppGpp pathway, how it affects bacterial growth, survival and virulence, and its connection with antibiotic tolerance and persistence. We will emphasize the dysfunctions of cells living without (p)ppGpp and finalize by reviewing the efforts and prospects of developing inhibitors of this pathway, and how these could be employed to improve current antibiotic therapy., Competing Interests: Competing interestsThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2021.)

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, a ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic interchromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. In addition, an Spx-independent pathway reduced the length of the cytokinetic period, perhaps by increasing divisome activity. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation. IMPORTANCE Bacteria must properly position the location of the cell division machinery in order to grow, divide, and ensure each daughter cell receives one copy of the chromosome. In Bacillus subtilis, cell division site selection depends on the Min and Noc systems, and while neither is individually essential, cells fail to grow at high temperature when both are mutated. Here, we show that cell division fails in the absence of Min and Noc, due not to a defect in FtsZ localization but rather to a failure in the maturation of the cell division machinery. Suppressor mutations that restored growth were selected, and while some activated the expression of ZapA via the Spx stress response pathway, others appeared to directly enhance divisome activity.

Bacteria that divide by binary fission form FtsZ rings at the geometric midpoint of the cell between the bulk of the replicated nucleoids. In Bacillus subtilis , the DNA- and membrane-binding Noc protein is thought to participate in nucleoid occlusion by preventing FtsZ rings from forming over the chromosome. To explore the role of Noc, we used time-lapse fluorescence microscopy to monitor FtsZ and the nucleoid of cells growing in microfluidic channels. Our data show that Noc does not prevent de novo FtsZ ring formation over the chromosome nor does Noc control cell division site selection. Instead, Noc corrals FtsZ at the cytokinetic ring and reduces migration of protofilaments over the chromosome to the future site of cell division. Moreover, we show that FtsZ protofilaments travel due to a local reduction in ZapA association, and the diffuse FtsZ rings observed in the Noc mutant can be suppressed by ZapA overexpression. Thus, Noc sterically hinders FtsZ migration away from the Z-ring during cytokinesis and retains FtsZ at the postdivisional polar site for full disassembly by the Min system. IMPORTANCE In bacteria, a condensed structure of FtsZ (Z-ring) recruits cell division machinery at the midcell, and Z-ring formation is discouraged over the chromosome by a poorly understood phenomenon called nucleoid occlusion. In B. subtilis , nucleoid occlusion has been reported to be mediated, at least in part, by the DNA-membrane bridging protein, Noc. Using time-lapse fluorescence microscopy of cells growing in microchannels, we show that Noc neither protects the chromosome from proximal Z-ring formation nor determines the future site of cell division. Rather, Noc plays a corralling role by preventing protofilaments from leaving a Z-ring undergoing cytokinesis and traveling over the nucleoid., (Copyright © 2021 Yu et al.)

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway., (© 2020 Sastre et al.)

Violacein is a tryptophan-derived purple pigment produced by environmental bacteria, which displays multiple biological activities, including strong inhibition of Gram-positive pathogens. Here, we applied a combination of experimental approaches to identify the mechanism by which violacein kills Gram-positive bacteria. Fluorescence microscopy showed that violacein quickly and dramatically permeabilizes B. subtilis and S. aureus cells. Cell permeabilization was accompanied by the appearance of visible discontinuities or rips in the cytoplasmic membrane, but it did not affect the cell wall. Using in vitro experiments, we showed that violacein binds directly to liposomes made with commercial and bacterial phospholipids and perturbs their structure and permeability. Furthermore, molecular dynamics simulations were employed to reveal how violacein inserts itself into lipid bilayers. Thus, our combined results demonstrate that the cytoplasmic membrane is the primary target of violacein in bacteria. The implications of this finding for the development of violacein as a therapeutic agent are discussed.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Magnetotactic bacteria, for the most part, are free-living, motile, unicellular prokaryotes that inhabit almost all marine and freshwater environments. One notable exception to the unicellular mode, however, are the magnetotactic multicellular prokaryotes. These morphologically unique prokaryotes (e.g., Candidatus Magnetoglobus multicellularis) are motile aggregates of 20-40 genetically identical, Gram-negative cells organised as a sphere (or ovoid in shape) and only motile as a unit. No specific close physical association between magnetotactic bacteria and non-magnetotactic microorganisms has ever been reported. Here, using culture-independent approaches, we show an unusual association between the spherical magnetotactic multicellular prokaryote Ca. Magnetoglobus multicellularis and Pseudoalteromonas species in environmental sediment and water samples collected from the Araruama Lagoon in Brazil. Cells of Pseudoalteromonas species were observed to be physically attached to the surface and, notably, even in the intercellular space of these spherical magnetotactic multicellular prokaryotes. An attempt to correlate the frequency of association between Pseudoalteromonas and magnetotactic multicellular prokaryotes with sediment depth was made but only a slight decrease in the number of Pseudoalteromonas cells per magnetotactic multicellular prokaryote was observed with increasing depth. Similar observations were made with magnetotactic multicellular prokaryotes from another Brazilian Lagoon (Rodrigo de Freitas) and the putative symbiont/parasite was detected. Although our results suggest some sort of specificity in the relationship between these prokaryotes, the precise nature of this association remains unclear.

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH 2 ), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100‑alanyl‑hexadecyl‑1‑amine (BP100-Ala-NH-C 16 H 33 ) and cyclo(1‑4)‑d‑Cys 1 , Ile 2 , Leu 3 , Cys 4 -BP100 (Cyclo(1‑4)‑cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C 16 H 33 acquired α-helical conformation, while Cyclo(1‑4)‑cILC-BP100) was partly α-helical and partly β-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram-negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram-positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp-synthetases (Rel Bs , RelP and RelQ) or bearing a Rel Bs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated., (© 2016 John Wiley & Sons Ltd.)

PlsX is a central enzyme of phospholipid synthesis in bacteria, converting acyl-ACP to acyl-phosphate on the pathway to phosphatidic acid formation. PlsX has received attention because it plays a key role in the coordination of fatty acid and phospholipid synthesis. Recently, PlsX was also suggested to coordinate membrane synthesis with cell division in Bacillus subtilis. Here, we have re-investigated the cell biology of PlsX and determined that the enzyme is uniformly distributed on the membrane of most cells, but occasionally appears as membrane foci as well. Foci and homogenous patterns seem freely interconvertible but the prevalence of the uniform staining suggests that PlsX does not need to localize to specific sites to function correctly. We also investigated the relationship between PlsX and the divisome. In contrast to previous observations, PlsX's foci showed no obvious periodicity of localization and did not colocalize with the divisome. Furthermore, depletion of PlsX did not affect cell division if phospholipid synthesis is maintained by an alternative enzyme. These results suggest that coordination between division and membrane synthesis may not require physical or functional interactions between the divisome and phospholipid synthesis enzymes., (© 2016 John Wiley & Sons Ltd.)

Bacterial division begins with the formation of a contractile protein ring at midcell, which constricts the bacterial envelope to generate two daughter cells. The central component of the division ring is FtsZ, a tubulin-like protein capable of self-assembling into filaments which further associate into a higher order structure known as the Z ring. Proteins that bind to FtsZ play a crucial role in the formation and regulation of the Z ring. One such protein is ZapA, a widely conserved 21 kDa homodimeric protein that associates with FtsZ filaments and promotes their bundling. Although ZapA was discovered more than a decade ago, the structural details of its interaction with FtsZ remain unknown. In this work, backbone and side chain NMR assignments for the Geobacillus stearothermophilus ZapA homodimer are described. We titrated FtsZ into (15)N(2)H-ZapA and mapped ZapA residues whose resonances are perturbed upon FtsZ binding. This information provides a structural understanding of the interaction between FtsZ and ZapA.

Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.

Bacterial cell division proteins must assemble at the middle of the cell to ensure the viability of both daughter cells. The first step in the assembly of the cell division apparatus is the polymerization of the tubulin-like protein FtsZ into a ring-shaped scaffold, the Z-ring. The Min system contributes to the spatial precision of division by inhibiting FtsZ polymerization at the cell poles. The component of this system that interacts with FtsZ is MinC, a 25 kDa protein that has two domains. The N-terminal domain of MinC is the main responsible for FtsZ inhibition, being sufficient to block Z-ring assembly when overexpressed in vivo, and to inhibit FtsZ polymerization in vitro. Despite intensive studies, little is known about the MinC binding site for FtsZ. We have assigned the backbone and side chain resonances of the MinC N-terminal domain of Bacillus subtilis through NMR spectroscopy. These assignments provide the basis to characterize the interaction between the N-terminal domain of MinC and FtsZ by NMR methods.

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.

ComN (YrzD) is a small, 98-amino-acid protein recently shown to be involved in the posttranscriptional control of the late competence comE operon in Bacillus subtilis. We show here that ComN localizes to the division site and cell poles in a DivIVA-dependent fashion. Yeast two-hybrid and glutathione S-transferase pulldown experiments showed that ComN interacts directly with DivIVA. ComN is not essential for the polar assembly of the core competence DNA uptake machinery. Nevertheless, polar localization of ComN should play some role in competence acquisition because delocalization of ComN leads to a small reduction in competence efficiency. We found that ComN promotes the accumulation of its target comE mRNA to septal and polar sites. Thus, we speculate that localized translation of ComE proteins may be required for efficient competence development. Our results underscore the versatility of DivIVA as a promoter of the differentiation of bacterial poles and demonstrate that the repertoire of polarly localized molecules in B. subtilis is broad, including a regulator of gene expression and its target mRNA. Moreover, our findings suggest that mRNA localization may play a role in the subcellular organization of bacteria.

Spore formation in Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of cell-cell communication. One pathway, which couples the proteolytic activation of the mother cell transcription factor σ(E) to the action of a forespore synthesized signal molecule, SpoIIR, has remained enigmatic. Signalling by SpoIIR requires the protein to be exported to the intermembrane space between forespore and mother cell, where it will interact with and activate the integral membrane protease SpoIIGA. Here we show that SpoIIR signal activity as well as the cleavage of its N-terminal extension is strictly dependent on the prespore fatty acid biosynthetic machinery. We also report that a conserved threonine residue (T27) in SpoIIR is required for processing, suggesting that signalling of SpoIIR is dependent on fatty acid synthesis probably because of acylation of T27. In addition, SpoIIR localization in the forespore septal membrane depends on the presence of SpoIIGA. The orchestration of σ(E) activation in the intercellular space by an acylated signal protein provides a new paradigm to ensure local transmission of a weak signal across the bilayer to control cell-cell communication during development., (© 2011 Blackwell Publishing Ltd.)

We describe AMIN (Amidase N-terminal domain), a novel protein domain found specifically in bacterial periplasmic proteins. AMIN domains are widely distributed among peptidoglycan hydrolases and transporter protein families. Based on experimental data, contextual information and phyletic profiles, we suggest that AMIN domains mediate the targeting of periplasmic or extracellular proteins to specific regions of the bacterial envelope.

Cell division in bacteria is carried out by an elaborate molecular machine composed of more than a dozen proteins and known as the divisome. Here we describe the characterization of a new divisome protein in Bacillus subtilis called YpsB. Sequence comparisons and phylogentic analysis demonstrated that YpsB is a paralog of the division site selection protein DivIVA. YpsB is present in several gram-positive bacteria and likely originated from the duplication of a DivIVA-like gene in the last common ancestor of bacteria of the orders Bacillales and Lactobacillales. We used green fluorescent protein microscopy to determine that YpsB localizes to the divisome. Similarly to that for DivIVA, the recruitment of YpsB to the divisome requires late division proteins and occurs significantly after Z-ring formation. In contrast to DivIVA, however, YpsB is not retained at the newly formed cell poles after septation. Deletion analysis suggests that the N terminus of YpsB is required to target the protein to the divisome. The high similarity between the N termini of YpsB and DivIVA suggests that the same region is involved in the targeting of DivIVA. YpsB is not essential for septum formation and does not appear to play a role in septum positioning. However, a ypsB deletion has a synthetic effect when combined with a mutation in the cell division gene ftsA. Thus, we conclude that YpsB is a novel B. subtilis cell division protein whose function has diverged from that of its paralog DivIVA.

FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (approximately 9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an approximately 3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, approximately 8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

Cell division in bacteria is mediated by the tubulin-like protein FtsZ, which assembles into a structure known as the Z ring at the future site of cytokinesis. We report the discovery of a Z-ring-associated protein in Bacillus subtilis called ZapA. ZapA was found to colocalize with the Z ring in vivo and was capable of binding to FtsZ and stimulating the formation of higher-order assemblies of the cytokinetic protein in vitro. The absence of ZapA alone did not impair cell viability, but the absence of ZapA in combination with the absence of a second, dispensable division protein EzrA caused a severe block in cytokinesis. The absence of ZapA also caused lethality in cells producing lower than normal levels of FtsZ or lacking the division-site-selection protein DivIVA. Conversely, overproduction of ZapA reversed the toxicity of excess levels of the division inhibitor MinD. In toto, the evidence indicates that ZapA is part of the cytokinetic machinery of the cell and acts by promoting Z-ring formation. Finally, ZapA is widely conserved among bacteria with apparent orthologs in many species, including Escherichia coli, in which the orthologous protein exhibited a strikingly similar pattern of subcellular localization to that of ZapA. Members of the ZapA family of proteins are likely to be a common feature of the cytokinetic machinery in bacteria.

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenotypical gentamicin resistance amplification (frequencies of 10(-3) to 10(-5), compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromosomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

Transposable elements of the mariner/Tc1 family are postulated to have spread by horizontal transfer and be relatively independent of host-specific factors. This was tested by introducing the Drosophila mauritiana element mariner into the human parasite Leishmania major, a trypanosomatid protozoan belonging to one of the most ancient eukaryotic lineages. Transposition in Leishmania was efficient, occurring in more than 20 percent of random transfectants, and proceeded by the same mechanism as in Drosophila. Insertional inactivation of a specific gene was obtained, and a modified mariner element was used to select for gene fusions, establishing mariner as a powerful genetic tool for Leishmania and other organisms. These experiments demonstrate the evolutionary range of mariner transposition in vivo and underscore the ability of this ubiquitous DNA to parasitize the eukaryotic genome.

The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, and cultured mammalian cells. Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus. First, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (+/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10(-4) to 10(-6) and was elevated 24-fold by treatment with gamma-irradiation, while the frequency of other events was less than 10(-6) and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the generation of both targeted and random mutations. We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by negative selection yielded parasites lacking DHFR-TS or foreign sequences. The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists. Such marker-free auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.

A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

A PCR based assay was designed in order to amplify putative ras gene sequences of the GTPase superfamily eventually present in Leishmania amazonensis and Trypanosoma cruzi. A set of primers corresponding to the conserved motifs G1 and G3 of the GTP binding proteins was synthesized. Sequencing of six PCR products (three from Leishmania and three from Trypanosoma) identified, however, two other different GTPases. The 270 bp L. amazonensis clone, pLef-11, shared an amino acid identity of around 80% with an eukaryotic elongation factor 1a of protein synthesis. On the other hand, the 168 bp T. cruzi clone, pTCr1, demonstrated over 60% amino acid identity to ras-related proteins of the rab-YPT-SEC4 family involved in control of vesicular traffic. To our knowledge, this is the first report of GTP binding protein genes in trypanosomatids.