A Look into the Melting Pot: The mecC-Harboring Region Is a Recombination Hot Spot in Staphylococcus stepanovicii

Introduction

Horizontal gene transfer (HGT) is an important driver for resistance- and virulence factor accumulation in pathogenic bacteria such as Staphylococcus aureus.

Methods

Here, we have investigated the downstream region of the bacterial chromosomal attachment site (attB) for the staphylococcal cassette chromosome mec (SCCmec) element of a commensal mecC-positive Staphylococcus stepanovicii strain (IMT28705; ODD4) with respect to genetic composition and indications of HGT. S. stepanovicii IMT28705 was isolated from a fecal sample of a trapped wild bank vole (Myodes glareolus) during a screening study (National Network on “Rodent-Borne Pathogens”) in Germany. Whole genome sequencing (WGS) of IMT28705 together with the mecC-negative type strain CM7717 was conducted in order to comparatively investigate the genomic region downstream of attB (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KR732654","term_id":"922979931"}}KR732654 and {"type":"entrez-nucleotide","attrs":{"text":"KR732653","term_id":"922979910"}}KR732653).

Results

The bank vole isolate (IMT28705) harbors a mecC gene which shares 99.2% nucleotide (and 98.5% amino acid) sequence identity with mecC of MRSA_LGA251. In addition, the mecC-encoding region harbors the typical blaZ-mecC-mecR1-mecI structure, corresponding with the class E mec complex. While the sequences downstream of attB in both S. stepanovicii isolates (IMT28705 and CM7717) are partitioned by 15 bp direct repeats, further comparison revealed a remarkable low concordance of gene content, indicating a chromosomal “hot spot” for foreign DNA integration and exchange.

Conclusion

Our data highlight the necessity for further research on transmission routes of resistance encoding factors from the environmental and wildlife resistome.     

Different Modes of Diaminopimelate Synthesis and Their Role in Cell Wall Integrity: a Study with Corynebacterium glutamicum

In eubacteria, there are three slightly different pathways for the synthesis of m-diaminopimelate (m-DAP), which is one of the key linking units of peptidoglycan. Surprisingly, for unknown reasons, some bacteria use two of these pathways together. An example is Corynebacterium glutamicum, which uses both the succinylase and dehydrogenase pathways for m-DAP synthesis. In this study, we clone dapD and prove by enzyme experiments that this gene encodes the succinylase (M_r = 24082), initiating the succinylase pathway of m-DAP synthesis. By using gene-directed mutation, dapD, as well as dapE encoding the desuccinylase, was inactivated, thereby forcing C. glutamicum to use only the dehydrogenase pathway of m-DAP synthesis. The mutants are unable to grow on organic nitrogen sources. When supplied with low ammonium concentrations but excess carbon, their morphology is radically altered and they are less resistant to mechanical stress than the wild type. Since the succinylase has a high affinity toward its substrate and uses glutamate as the nitrogen donor, while the dehydrogenase has a low affinity and incorporates ammonium directly, the m-DAP synthesis is another example of twin activities present in bacteria for access to important metabolites such as the well-known twin activities for the synthesis of glutamate or for the uptake of potassium.     

Remote stimulation by heat induces characteristic membrane-potential responses in the veins of wild-type and abscisic acid-deficient tomato plants

The systemic induction of proteinase inhibitor genes in tomato plants is either mediated by fast electrical signals or alternatively by chemical messengers. In the present study we analyzed the pathway of the electrical signal. The question of which cell types are involved in this pathway of long-distance signaling within plants is still controversial. To identify these we inserted microelectrodes into the veins of tomato leaves (Lycopersicon esculentum Mill. cv. Moneymaker). A newly developed computer program and microcomputer interface enabled us to position these microelectrodes inside the vein with an accuracy of 1 μm. Due to this precision in positioning we were able to demonstrate that the pathway of the electrical signal is not restricted to a specific tissue type, e.g. the phloem. In particular, the entire vein contributes to the propagation of the electrical wave along the plant. Therefore, an apoplastic contribution to the long-distance signal transduction mechanism appears most likely. To furthermore investigate the involvement of cis-abscisic acid (ABA) in this long-distance signal transduction pathway, ABA-deficient tomato mutants (Lycopersicon esculentum cv. Sitiens) were used in comparison to the wild type. Significant differences between the membrane-potential relaxation kinetics of the wild type and the mutants could be detected. Wild-type tomato plants exhibited six characteristic classes of membrane-potential relaxation kinetics following heat treatment. In contrast, the ABA-deficient mutants were more restricted in terms of their relaxation upon heat stimulation. The responses in the membrane potential of all cells within a vein consisted of only three categories. In conclusion, ABA did not affect all cells within the vein in a similar manner. Single cells exhibited different response patterns to systemic heat application in the presence of ABA. Moreover, ABA had a pronounced effect on the resting potentials of individual cells within the veins of tomato. Received: 1 July 1997 / Accepted: 16 January 1998     

Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Aspirin prevents wound-induced gene expression in tomato leaves by blocking jasmonic acid biosynthesis

Jasmonic acid (JA) and its methyl ester, like mechanical wounding, strongly induce accumulation of proteinase inhibitor II (Pin2) in tomato and potato leaves. In plants, JA is synthesized from α-linolenic acid by a lipoxygenase (LOX)-mediated oxygenation leading to 13-hydroxyperoxylinolenic acid (13-HPLA) which is then subsequently transformed to JA by the action of hydroperoxide-dehydrase activity and additional modification steps. Both the chemical structure as well as the biosynthetic pathway of JA resemble those of the mammalian eicosanoids (prostaglandins and leukotrienes) which are derived from LOX-and cyclooxygenase (COX)-mediated reactions. To assess the role of endogenous JA in the wound response, detached tomato (Lycopersicon esculentum Mill.) leaves were supplied with different LOX and COX inhibitors and the expression of the wound-induced genes for Pin2 (Pin2), cathepsin D inhibitor (Cdi) and threonine deaminase (Td) was analyzed. Lipoxygenase inhibitors as well as some COX inhibitors blocked the wound-induced accumulation of Pin2, Cdi and Td mRNA. Quantitation of endogenous levels of JA showed that aspirin blocks the increase of this phytohormone normally observed as a result of wounding. Linolenic acid and 13-HPLA do not induce the expression of Pin2, Cdi and Td in the presence of aspirin. However, 12-oxo-phytodienoic acid and jasmonic acid are able to overcome the inhibitory effect of this substance. These results strongly indicate that aspirin prevents wound-induced gene activation by inhibiting the hydroxyperoxide-dehydrase activity that mediates the conversion of 13-HPLA to 12-oxo-phytodienoic acid.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Erratum

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco