Discovery of a Diverse Set of Bacteria That Build Their Cell Walls without the Canonical Peptidoglycan Polymerase aPBP.

Peptidoglycan (PG) is a highly cross-linked peptide-glycan mesh that confers structural rigidity and shape to most bacterial cells. Polymerization of new PG is usually achieved by the concerted activity of two membrane-bound machineries, class-A penicillin binding proteins (aPBPs) and class-B penicillin binding proteins (bPBPs) in complex with shape, elongation, division, and sporulation (SEDS) proteins. Here, we have identified four phylogenetically distinct groups of bacteria that lack any identifiable aPBPs. We performed experiments on a panel of species within one of these groups, the Rickettsiales, and found that bacteria lacking aPBPs build a PG-like cell wall with minimal abundance and rigidity relative to cell walls of aPBP-containing bacteria. This reduced cell wall may have evolved to minimize the activation of host responses to pathogens and endosymbionts while retaining the minimal PG-biosynthesis machinery required for cell elongation and division. We term these "peptidoglycan-intermediate" bacteria, a cohort of host-associated species that includes some human pathogens. IMPORTANCE Peptidoglycan (PG) is a large, cross-linked polymer that forms the cell wall of most bacterial species and confers shape, rigidity, and protection from osmotic shock. It is also a potent stimulator of the immune response in animals. PG is normally polymerized by two groups of enzymes, aPBPs and bPBPs working together with shape, elongation, division, and sporulation (SEDS) proteins. We have identified a diverse set of host-associated bacteria that have selectively lost aPBP genes while retaining bPBP/SEDS and show that some of these build a minimal PG-like structure. It is expected that these minimal cell walls built in the absence of aPBPs improve the evolutionary fitness of host-associated bacteria, potentially through evasion of PG-recognition by the host immune system.

Diversity and metabolic potential of the microbiota associated with a soil arthropod.

Springtails are important members of the soil fauna and play a key role in plant litter decomposition, for example through stimulation of the microbial activity. However, their interaction with soil microorganisms remains poorly understood and it is unclear which microorganisms are associated to the springtail (endo) microbiota. Therefore, we assessed the structure of the microbiota of the springtail Orchesella cincta (L.) using 16S rRNA gene amplicon sequencing. Individuals were sampled across sites in the field and the microbiota and in particular the endomicrobiota were investigated. The microbiota was dominated by the families of Rickettsiaceae, Enterobacteriaceae and Comamonadaceae and at the genus level the most abundant genera included Rickettsia, Chryseobacterium, Pseudomonas, and Stenotrophomonas. Microbial communities were distinct for the interior of the springtails for measures of community diversity and exhibited structure according to collection sites. Functional analysis of the springtail bacterial community suggests that abundant members of the microbiota may be associated with metabolism including decomposition processes. Together these results add to the understanding of the microbiota of springtails and interaction with soil microorganisms including their putative functional roles.

Evidence for a peptidoglycan-like structure in Orientia tsutsugamushi.

Bacterial cell walls are composed of the large cross-linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan-like structure in Orientia, as well as an outer membrane containing a network of cross-linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.

Ixodes ricinus and Its Endosymbiont Midichloria mitochondrii: A Comparative Proteomic Analysis of Salivary Glands and Ovaries.

Hard ticks are hematophagous arthropods that act as vectors of numerous pathogenic microorganisms of high relevance in human and veterinary medicine. Ixodes ricinus is one of the most important tick species in Europe, due to its role of vector of pathogenic bacteria such as Borrelia burgdorferi and Anaplasma phagocytophilum, of viruses such as tick borne encephalitis virus and of protozoans as Babesia spp. In addition to these pathogens, I. ricinus harbors a symbiotic bacterium, Midichloria mitochondrii. This is the dominant bacteria associated to I. ricinus, but its biological role is not yet understood. Most M. mitochondrii symbionts are localized in the tick ovaries, and they are transmitted to the progeny. M. mitochondrii bacteria have however also been detected in the salivary glands and saliva of I. ricinus, as well as in the blood of vertebrate hosts of the tick, prompting the hypothesis of an infectious role of this bacterium. To investigate, from a proteomic point of view, the tick I. ricinus and its symbiont, we generated the protein profile of the ovary tissue (OT) and of salivary glands (SG) of adult females of this tick species. To compare the OT and SG profiles, 2-DE profiling followed by LC-MS/MS protein identification were performed. We detected 21 spots showing significant differences in the relative abundance between the OT and SG, ten of which showed 4- to 18-fold increase/decrease in density. This work allowed to establish a method to characterize the proteome of I. ricinus, and to detect multiple proteins that exhibit a differential expression profile in OT and SG. Additionally, we were able to use an immunoproteomic approach to detect a protein from the symbiont. Finally, the method here developed will pave the way for future studies on the proteomics of I. ricinus, with the goals of better understanding the biology of this vector and of its symbiont M. mitochondrii.

Rickettsiaceae, rickettsia-like endosymbionts, and the origin of mitochondria.

Accumulating evolutionary data point to a monophyletic origin of mitochondria from the order Rickettsiales. This large group of obligate intracellular alpha-Proteobacteria includes the family Rickettsiaceae and several rickettsia-like endosymbionts (RLEs). Detailed phylogenetic analysis of small subunit (SSU) rRNA and chaperonin 60 (Cpn60) sequences testify to polyphyly of the Rickettsiales, and consistently indicate a sisterhood of Rickettsiaceae and mitochondria that excludes RLEs. Thus RLEs are considered as the nearest extant relatives of an extinct last common ancestor of mitochondria and rickettsiae. Phylogenetic inferences prompt the following assumptions. (1) Mitochondrial origin has been predisposed by the long-term endosymbiotic relationship between rickettsia-like bacteria and proto-eukaryotes, in which many endosymbiont genes have been lost while some indispensable genes have been transferred to the host genome. (2) The obligate dependence of rickettsiae upon a eukaryotic host rests on the import of proteins encoded by these transferred genes. The nature of a proto-eukaryotic cell still remains elusive. The divergence of Rickettsiaceae and mitochondria based on Cpn60, and the evolutionary history of two aminoacyl-tRNA synthetases favor the hypothesis that it was a chimera created by fusion of an archaebacterium and a eubacterium not long before an endosymbiotic event. These and other, mostly biochemical data suggest that all the mitochondrion-related organelles, i.e., both aerobically and anaerobically respiring mitochondria and hydrogenosomes, have originated from the same RLE, while hydrogenosomal energy metabolism may have a separate origin resulting from a eubacterial fusion partner.

Reproductive characteristics and strategies of reducing-system bivalves.

The reproductive biology of Type 3 reducing-system bivalves (those whose pallial cavity is irrigated with water rich in reducing substances) is reviewed, with respect to size-at-maturity, sexuality, reproductive cycle, gamete size, symbiont transmission, and larval development/dispersal strategies. The pattern which emerges from the fragmentary data is that these organisms present reproductive particularities associated with their habitat, and with their degree of reliance on bacterial endosymbionts. A partial exception to this pattern is the genus Bathymodiolus, which also presents fewer trophic adaptations to the reducing environment, suggesting a bivalent adaptive strategy. A more complete understanding of the reproductive biology of Type 3 bivalves requires much more data, which may not be feasible for some aspects in the deep-sea species.

Analysis of Wolbachia protein synthesis in Drosophila in vivo.

Intracellular Wolbachia infections are extremely common in arthropods and exert profound control over the reproductive biology of the host. However, very little is known about the underlying molecular mechanisms which mediate these interactions with the host. We examined protein synthesis by Wolbachia in a Drosophila host in vivo by selective metabolic labelling of prokaryotic proteins and subsequent analysis by 1D and 2D gel electrophoresis. Using this method we could identify the major proteins synthesized by Wolbachia in ovaries and testes of flies. Of these proteins the most abundant was of low molecular weight and showed size variation between Wolbachia strains which correlated with the reproductive phenotype they generated in flies. Using the gel systems we employed it was not possible to identify any proteins of Wolbachia origin in the mature sperm cells of infected flies.

Detection of Bartonella (Rochalimaea) quintana by routine acridine orange staining of broth blood cultures.

Bartonella quintana was isolated from 34 BACTEC nonradiometric aerobic resin blood cultures for 10 adults. Nine patients were initially diagnosed by routine acridine orange staining of routine cultures that had been incubated for 8 days. All subcultures grew on chocolate agar within 3 to 12 days (median, 6 days). The PLUS 26 high-volume aerobic resin medium, combined with acridine orange stain and subculture, is an effective system for detection and isolation of B. quintana from blood.

Proposed tests for the routine identification of Rochalimaea species.

A study was conducted to establish tests for the routine identification of Rochalimaea species. Strains used were reference strains of Rochalimaea vinsonii and Rochalimaea quintana, and a type strain and six human isolates of Rochalimaea henselae. Rochalimaea species were confirmed to be gram-negative, oxidase-negative, non-motile, urease-negative, indole-negative, catalase-negative, glucose-nonfermenting organisms which failed to grow on MacConkey agar. Further testing of the organisms in a commercial identification system with the addition of hemin (100 micrograms/ml) to the medium revealed biochemical reactivity of the organisms not previously observed. The Voges-Proskauer reaction, tests for hydrolysis of hippurate and esculin, leucine arylamidase activity and the lactose test allowed identification and differentiation of the three species. Rochalimaea henselae was the only species with a positive lactose test and Rochalimaea quintana was the only species with a positive Voges-Proskauer reaction. Further studies are needed to confirm the validity of these tests for identification of Rochalimaea species.

Biology of ehrlichiae.

The isolation and cultivation of the agent of Potomac horse fever, Ehrlichia risticii, by Holland, Ristic, et al., afforded Weisburg et al. an opportunity to examine its phylogeny. E. risticii is clearly related to the genus Rickettsia and not to chlamydiae. A reevaluation of the significance of phenotypic characteristics is thus required, since ehrlichiae, in some respects, resemble chlamydiae. For example, unlike rickettsiae, ehrlichiae and chlamydiae multiply in the phagosome of their host cells, but may not have the same mechanism of inhibition of phagosome-lyososome fusion. Rickettsiae, which multiply in the cytoplasm, may have a mechanism of survival in the phagosome similar to that of the ehrlichiae, but, in addition, utilize a phospholipase, which permits prompt escape from the phagosome. Rickettsiae, as most Gram negatives, multiply by binary fisson. Chlamydiae, on the other hand, undergo a cycle of development. Elementary bodies (EB) infect, but do not divide, while the reverse is true of reticulate bodies (RB). Ehrlichiae superficially resemble chlamydiae rather than rickettsiae, but ehrlichiae have not yet been submitted to the rigorous criteria of separation of EB and RB. Investigations in our laboratory of the metabolic activities of E. risticii and E. sennetsu link them to the rickettsiae and not to the chlamydiae. Ehrlichiae and rickettsiae, but not chlamydiae, derive some ATP from their catabolic activities. In conclusion, in further investigations of the monocytic ehrlichiae, it is safer to be guided by what we know of rickettsiae, than chlamydiae.

Energy metabolism of monocytic Ehrlichia.

We investigated if the monocytic Ehrlichia are totally dependent on their host cells for energy, or, as Rickettsia, are capable of some ATP synthesis in vitro. The Miyayama strain of Ehrlichia sennetsu and the Maryland and Illinois strains of Ehrlichia risticii were cultivated in a mouse macrophage cell line, separated from host cell constituents by procedures that included Renografin or Percoll gradient centrifugation, and tested after cryopreservation. Cells incubated without a metabolizing substrate contained little, if any, ATP. When the Ehrlichia cells were incubated for 1 hr at 34 degrees C with glutamine, significant amounts of ATP were detected. The amounts of ATP attained with glutamine were decreased in some instances by the addition of atractyloside, an inhibitor of adenine nucleotide translocase in mitochondria, and were decreased consistently and to a greater extent by 2,4-dinitrophenol. When ATP, instead of glutamine, was added to the ehrlichiae, upon incubation the amount of ATP was markedly decreased. Comparable responses under all these conditions were obtained with Rickettsia typhi, although the final ATP levels were higher. Control preparations derived from uninfected mouse macrophages or from the discards of the Ehrlichia purification procedures contained negligible amounts of ATP, which were not increased by incubation with glutamine. We conclude that with respect to ATP metabolism, the monocytic Ehrlichia resemble Rickettsia more closely than Chlamydia, even though Ehrlichia resemble Chlamydia in their intracellular location in the phagosomes and in possibly having a developmental cycle.

Substrate utilization by Ehrlichia sennetsu and Ehrlichia risticii separated from host constituents by renografin gradient centrifugation.

The in vitro metabolic activities of two monocytic species of Ehrlichia were investigated. The Miyayama strain of Ehrlichia sennetsu and two strains of Ehrlichia risticii, isolated in Illinois and Maryland, were cultivated in a P388D1 mouse macrophage cell line. The ehrlichia particles from heavily infected cultures were separated from host constituents by a Renografin gradient centrifugation procedure modified from those employed for rickettsiae and chlamydiae. The metabolic activities of the isolated ehrlichiae were measured by their formation of CO2 after incubation for 1 h or longer at 34 degrees C with 14C-labeled substrates. Of the substrates tested, glutamine was utilized most vigorously. The greatest activity was obtained at pH 7.2 to 8.0, while the activity rapidly declined at pH below 7. The most favorable buffer was one that contained 0.05 M potassium phosphate as well as 0.2 M sucrose, thus affording some osmotic protection. Glutamate was utilized to a much lesser extent than glutamine, and glucose was not utilized at all. No consistent differences in metabolic activities among the three strains were observed.

Metabolism and genetics of chlamydias and rickettsias.

Chlamydial and rickettsial diseases pose a hazard to man and to domesticated and wild animals. The virulence mechanisms which aid the establishment of these obligate intracellular parasites in the eukaryotic host are still not within our grasp. Recent knowledge of the biochemical stratagem, the metabolic capabilities and the genetic diversity of these microbes illustrate fundamental differences in ecology and evolutionary divergence. The preferred site of intracellular residence determines the strategy for uptake, for nutrient assimilation and also for evasion of the host's immunological defenses. The Chlamydia, Rickettsia, and Coxiella are the most extensively studied of the genera. Whereas the Ehrlichia and Cowdria are poorly understood, they are also the most intriguing of the Rickettsiae. A number of antigenically and genetically distinct species are identified for the genera Chlamydia, Rickettsia, and Ehrlichia, whereas the Coxiella and Cowdria may not represent such a wide diversity. Recent information on the genetic heterogeneity of the chromosomal and plasmid DNAs of the strains of Coxiella suggest the diversity is greater than was originally envisioned. New information regarding the antigenic structure of Cowdria and their cellular tropisms suggests that they are closely related to the Ehrlichia. In this review we compare the metabolic capabilities and the genetic diversity of these different intracellular bacteria.

Biochemistry of rickettsiae: recent advances.

The application of new biotechnology to the study of the biochemistry of rickettsiae was a prominent feature of the presentations at the 3rd International Symposium on Rickettsiae and Rickettsial Diseases, held in Smolenice near Bratislava in September 1984. This review is an attempt to summarize recent advances leading up to these presentations as well as the studies that have been reported in the two years since the meeting. Since rickettsiae are intracellular parasites, most reviews deal with the interaction of rickettsiae with host cells. It is useful, however, to focus also--as we have done--on the properties of rickettsiae that can be demonstrated in the absence of their hosts, although, undoubtedly, many of these properties reflect adaptation to an intracellular microenvironment.

Protein synthesis by intracellular symbionts in two closely interrelated aphid species.

An aphid endosymbiont in vivo synthesizes symbionin almost exclusively which is not produced in vitro by the same symbiont. While symbionin produced by the endosymbiont of the pea aphid is an acidic protein with a molecular weight of 63,000, that by the symbiont of the kondo aphid, the closest relative to the former, is a distinct, less acidic, molecule. While the two endosymbionts in vivo in old insects synthesize about 11 protein species in common, they produce many different proteins when incubated extracellularly.

Ornithine metabolism in the genus Rochalimaea.

Ornithine metabolism was studied in two strains of the trench fever rickettsia Rochalimaea quintana, Fuller and Guadalupe, and in the vole agent, a strain of Rochalimaea but not necessarily of Rochalimaea quintana. The metabolic activity of intact cells and cell-free extracts was measured by monitoring the evolution of 14CO2 from [1-14C]ornithine. Low levels of activity were obtained with all three strains, but requirements for the demonstration of this activity differed. With the cells of the Fuller and Guadalupe strains, the decarboxylation of ornithine was almost completely dependent on added pyruvate or succinate, presumably as sources of energy for transport. This enhancement was not prevented by the presence of chloramphenicol. The activity of the vole agent, on the other hand, required the complete medium. This activity was prevented by chloramphenicol added at the same time as the medium but not by chloramphenicol added after 1 h of incubation. In cell-free extracts, the demonstration of ornithine decarboxylase activity in the vole agent required prior induction with medium containing ornithine, whereas in the other two strains, the activity was constitutive. The activities of the extracts of the Fuller strain and the vole agent differed also in pH optimum, which was somewhat lower for the vole agent, and in the added pyridoxal phosphate requirement, which was greater for the Fuller strain. Comparable experiments with Rickettsia typhi and Rickettsia prowazekii failed to reveal evidence of ornithine metabolism.

[Oscillations in the energy metabolism of the host and symbiont of a cicada. I. Analysis of possible metabolic and physiologic correlations of both systems]. / Oszillationen im Energiestoffwechsel von Wirt und Symbiont eines Zikadeneis. I. Analyse möglicher stoffwechselphysiologischer Korrelationen beider Systeme.

The insect group which includes cicadas harbours intracellular bacterial symbionts which are passed on from generation to generation in the form of a 'symbiont ball' inserted between the egg membrane and the rear pole of the egg cell. Bioluminiscence methods can be used to measure the oscillations in ATP, ADP and AMP levels in egg systems which have been separated into a host and a symbiont part (Euscelidius variegatus, Euscelis incisus), and which are exposed to constant light or light-dark variations under otherwise constant conditions (26 degrees C, 70% relative humidity, 7000 lux). The energy charge can be calculated from the ATP, ADP and AMP concentrations. Comparisons of such curves suggest an 'antagonistic' relationship in the energy metabolism of the host and symbiont parts of the egg. The minima of oscilaltions in the host's energy metabolism generally occur at the same time as the maxima in the endocytobionts energy metabolism. Antagonistic correlations between the nucleus/cytoplasm and mitochondria/plastids were also observed in the eucyte system. Analogies between the two systems can be explained satisfactorily by the endosymbiont theory of the origin of eucytes. It follows that insect endocytobioses can serve as an experimental model for the biochemical analysis of the eucyte system.

Vole agent identified as a strain of the trench fever rickettsia, Rochalimaea quintana.

The vole agent described by Baker in 1946 was studied as an example of a bacterium that has been mistakenly regarded a rickettsia. Unlike rickettsiae, the vole agent killed chicken embryos with great irregularity, multipled primarily at the surface of avian or mammalian cells and not intracellularly, produced colonies rather than plaques on chicken embryo monolayers under agar, and developed small colonies after 4 to 7 days of cultivation on blood plates. It was most conveniently cultivated on monolayers of irradiated L cells and was purified by minor modifications of the Renografin gradient procedure used for rickettsiae. It actively catabolized glutamate, glutamine, succinate, and pyruvate, but not glucose or glucose-6-phosphate. Enzymatic activities of cell extracts were consistent with above findings. The base ratio (molar percent guanine plus cytosine) of its deoxyribonucleic acid was shown to be 39, which was identical to the base ratio of the deoxyribonucleic acid of Rochalimaea quintana tested simultaneously. Serological studies indicated no cross-reactivity with Rickettsia tsutsugamushi, but strong cross-reaction with R. quintana was observed when a hyperimmune rabbit serum and a convalescent human serum were tested. We conclude that the vole agent is a strain of the trench fever rickettsia, R. quintana.