Xcp-mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression

In Pseudomonas aeruginosa, several exoproteins synthesized with a signal sequence (elastase, lipase, phospholipases, alkaline phosphatase and exotoxin A) are secreted by a two-step mechanism. They first cross the inner membrane in a signal sequence-dependent way, and are further translocated across the outer membrane in a second step requiring secretion functions encoded by several xcp genes. Ten xcp genes have already been characterized (Bally et al., 1992a). In this study, two additional xcp genes, xcpP and xcpQ, are described. They are located in the 40 min region of the chromosome where they probably define an operon, divergent from the xcpR–Z operon previously characterized in this region. These two genes encode two proteins, XcpP and XcpQ, similar to PulC and PulD of the pul system of Klebsiella oxytoca. Moreover, the two divergent operons share a common regulation which is growth-phase dependent.     

Composite origin of major histocompatibility complex genes

Major histocompatibility complex WHO genes have now been cloned from representatives of all vertebrate classes except Agnatha. The recent accumulation of sequence data has given great insight into the course of evolution of these genes. Although the primary structure of the MHC genes varies greatly from class to class and also within the individual classes, the general features of the tertiary and quaternary structure have been conserved remarkably well during more than 400 million years.of evolution. The ancestral MHC genes may have been assembled from at least three structural elements derived from different gene families. Class II MHC genes appear to have been assembled first, and then to have given rise to class I genes.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

Cloning and characterization of a new polyketide synthase gene cluster in Streptomyces aureofaciens CCM 3239.

We cloned a new polyketide gene cluster, aur2, in Streptomyces aureofaciens CCM3239. Sequence analysis of the 9531-bp DNA fragment revealed 10 open reading frames, majority of which showed high similarity to the previously characterized type II polyketide synthase (PKS) genes. An unusual feature of the aur2 cluster is a disconnected organization of minimal PKS genes; ACP is located apart from the genes for ketosynthases KSalpha and KSbeta. The aur2 gene cluster was disrupted in S. aureofaciens CCM3239 by a homologous recombination, replacing the four genes (aur2A, E, F, G) including ketosynthase KSalpha, with antibiotic resistance marker gene. The disruption did not affect growth and differentiation, and disrupted strain produced spores with wild-type grey-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild type and aur2-disrupted strains did not reveal any difference in the pattern of antibacterial compounds.     

Social Behaviour of the Carpenter Bee Xylocopa pubescens (Spinola)

The development of about 20 relatively small nests of Xylocopa pubescens was studied. After the first offspring had become adult, these nests reached a social stage in which there was only one egg-layer per nest. Freshly emerged (teneral) adults eat a lot of food, collected by a forager, before they fly out of the nest. This food appears to be of major importance to these bees in that it makes them fully agile. It seems therefore, that part of the food needed for the development of a young bee is not given at the larval stage, via the beebread, but at the teneral adult stage. As a result of this, a necessary overlap of generations is accomplished. Besides, less pollen has to be collected for the provision of a brood cell, so more cells can be constructed within a short period of time. There is a high degree of nest competition. Many nests were taken over by conspecific individuals (or by females of X. sulcatipes) that were searching for nesting sites within the study area. However, although more than 50% of the solitary nests were taken over sooner or later, strangers hardly ever intruded into social nests, with more than one adult. This illustrates how important it is for a reproducing female to tolerate the presence of nestmates which guard the nest in her absence. Although in most cases the foundress of a nest proceeded to produce new brood in the presence of her offspring, it happened that a daughter took over reproduction from her mother. In two of these cases, it could be observed that a mother started a new nest elsewhere after having been thrown out by her own daughter. At least in these cases, nest competition between mother and daughter started long before the mother had reached the end of her reproductive capacities. Since nesting possibilities are scarce, it seems a logical strategy to stay in the maternal nest and wait for a chance to become the egg-layer if the mother dies or if she loses her dominance. The occurrence of several social interactions among nestmates is discussed: — trophallaxis was observed often, not only under ‘forced’ conditions, but also as a form of ‘voluntary’ feeding; — nestmates were observed to groom each other.     

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker ( ermC ), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)₂–(Hep)₂. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N -acetylglucosamine to heptose.