A protein critical for cell constriction in the Gram‐negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan‐binding LysM domains

During division of Gram‐negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast‐growth conditions. Under slow‐growth conditions, the ΔdipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non‐canonical. DipM also carries two tandems of the PG‐binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non‐septal, monolayered PG.     

A Cytohesin Homolog in Dictyostelium Amoebae

Background

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.

Principal Findings

Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG⁻ cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced.

Significance

The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.     

Single strand conformation polymorphism of selected genes of the Klebsiella pneumoniae cluster waa for core lipopolysaccharide biosynthesis

We aimed to determine single strand conformation polymorphism (SSCP) of selected waa cluster genes (waaA, waaE, waaL, waaQ and waaZ) involved in core lipopolysaccharide (LPS) synthesis in reference and epidemic strains of Klebsiella pneumoniae. Number of 24 reference strains belonging to serogroups O1, O2a, O2a2e, O2a2e2h, O2a2f2g, O3, O4, O5, O7, O8, and O12 was tested together with 13 epidemic strains from 5 outbreaks and 6 casual isolates using PCR and Multitemperature-SSCP. Based on PCR-SSCP results, from 4 to 8 patterns (genotypes) were distinguished for each analysed gene. Predomination of single genotype ranging from 28% to 76% for waaL and waaE respectivelly was observed in tested strains. The average predomination for other genes was about 36%. Although no correlation was observed between genotypes and serogroup of tested strains, it is notheworthy that epidemiologically linked isolates belonged to the same genotype. Therefore reported here heterogeneity of tested genes may be potentially useful for K. pneumoniae strains subtyping by SSCP or DNA sequencing.     

The molecular characteristic of Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical sources

The aim of study was the molecular characteristic of S. aureus and S. epidermidis isolates obtained from skin surface, wounds, deep tissues of hospitalized patients and from skin surface of non-hospitalized patients. Genes encoding virulence factors were examined using PCR reaction and specific primers. Genes encoding adhesinsfnbA and cna and gene eta for epidermolytic toxin were mostly present in S. aureus isolates coming from wounds and deep tissues compared to these from skin surface. Gene atlE encoding autolysin of S. epidermidis was detected in all studied isolates, whereas gene icaAB was present in almost all isolates. Comparison of results obtained by PCR and conventional method of the resistance to methicillin estimation showed discrepances suggesting the need for using of both methods in some clinically difficult cases of S. aureus infection.     

A comparative study of water distribution, free radical production and activation of antioxidative metabolism in germinating pea seeds

The aim of this study was to investigate whether there is a relationship between hydration of the embryo axes and cotyledons and the resumption of the oxidative metabolism in both organs of germinating seeds of pea (Pisum sativum L. cv. Piast). Nuclear magnetic resonance (¹H-NMR) spectroscopy and imaging were used to study temporal and spatial water uptake and distribution in pea seeds. The observations revealed that water penetrates into the seed through the hilum, micropyle and embryo axes, and cotyledons hydrate to different extents. Thus, inhomogeneous water distribution may influence the resumption of oxidative metabolism. Electron paramagnetic resonance (EPR) measurements showed that seed germination was accompanied by the generation of free radicals with g₁ and g₂ values of 2.0032 and 2.0052, respectively. The values of spectroscopic splitting coefficients suggest that they are quinone radicals. The highest content of free radicals was observed in embryo axes immediately after emergence of the radicle. Glutathione content decreased during the entire germination period in both embryo axes and cotyledons. A different profile was observed for ascorbate, with significant increases in embryo axes, coinciding with radicle protrusion. Electrophoretic analysis showed that superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) were present in dry seeds and were activated later during germination, especially in embryo axes. The presence of all antioxidative enzymes as well as low molecular antioxidants in dry seeds allowed the antioxidative machinery to be active as soon as the enzymes were reactivated by seed imbibition. The observed changes in free radical levels, antioxidant contents and enzymatic activities in embryo axes and cotyledons appear to be more closely related to metabolic and developmental processes associated with preparation for germination, and do not correspond directly to the hydration of the tissues.     

Maintaining proteostasis under mechanical stress

Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force‐unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.     

Changes in the nucleotide pools in leaves and buds of tobacco plants upon treatment with 2,4-dichlorophenoxyacetic acid

Tobacco ( Nicotiana tabacum L. cv. Samsun) plants were treated once with 2,4-dichlorophenoxyacetic acid (2,4-D) at the 8-leaf stage. The effect of the herbicide on leaf metabolism was followed over 7 days by determination of the ribonucleotide pools, including NAD⁺, NADP⁺ and UDP-sugars, by high-preformance liquid chromatography. 2,4-D treatment resulted in large changes in the nucleotide concentrations, the magnitude and sign of which were dependent upon the leafage. The nucleotide pools decreased in the apical tissue, but increased strongly in the mature leaves with the highest relative increase in the oldest leaf tested. The time course of the changes revealed a maximum on day 5 after 2,4-D treatment. The increase in the adenine nucleotide pools, energy charge and the NADVNADP⁺ ratio are interpreted to indicate a stress situation. The different responses of young, mature and senescent tissue to the synthetic auxin could reflect their different inherent sensitivity due to the natural auxin gradient.     

Sterols and sterylglycosides of oats (Avena sativa). Distribution in the leaf tissue and medium-induced glycosylation of sterols during protoplast isolation

Sterols, sterylglycosides (SG), acylated sterylglycosides (ASG) and steroidal saponins of primary leaves of oat ( Avena sativa L. cv. Flämingskrone) were analyzed by thin-layer chromatography, gas-liquid chromatography and high-performance liquid chromatography. Intact leaves, epidermis preparations, epidermis-stripped leaves, isolated protoplasts and chloroplasts were compared. The mesophyll contained 79% of the total leaf sterols, 80% of the SG and 78% of the ASG, but only 33–67% of the saponins. Free sterols, SG and ASG were mainly localized within the mesophyll, whereas steroidal saponins were localized in the epidermis to a significantly higher extent. The sterol parts consisted mainly of sitosterol, stigmasterol. cholesterol. Δ⁵-avenasterol, Δ⁷-avenasterol, campesterol and Δ⁷-cholestenol, and were quantitatively different in different sterol groups. A higher percentage of sitosterol at the expense of stigmasterol was typical for SG and ASG as compared to free sterols. Only minor differences in the sterol composition were found in a given sterol group when isolated from different tissues. Isolated protoplasts contained only 5–9% of the sterols present in mesophyll cells, indicating that the major part of the free sterols was lost during isolation. Exposure of radioactively labelled leaf segments to either buffer or digestion medium induced rapid transformation of sterols to SG and ASG as shown by the shift of radioactivity from free sterols to the glyeosides. This suggests that two sterol pools exist in the cell: one in the plasmalemma, which is accessible to medium-induced transformation, and a second non-accessible pool in the interior membranes (e.g. chloroplasts) of the cell.