Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis.

We report the discovery of a bacterial gene, tlpA, that codes for a hitherto unknown type of thioredoxin-like protein. The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nodule symbiont Bradyrhizobium japonicum. The TlpA protein shared up to 31% amino acid sequence identity with various eukaryotic and prokaryotic thioredoxins and protein disulfide isomerases, and possessed a characteristic active-site sequence, Trp-Cys-Val-Pro-Cys. In contrast to all members of the thioredoxin family known to date, TlpA was shown to be anchored to the cytoplasmic membrane by means of an N-terminal transmembrane domain, while the active site-containing part of the protein faced the periplasm. The tlpA mutant had a pleiotropic phenotype in that it was defective in the development of a nitrogen fixing endosymbiosis and exhibited a strongly decreased oxidase activity, as compared with the wild-type. Holocytochrome aa3 was spectroscopically undetectable in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic membrane. Since cytochrome aa3 is not a prerequisite for the development of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential periplasmic step in the maturation of cytochrome aa3.     

The refined crystal structure of ribonuclease A at 2.0 A resolution

This paper describes the structure of bovine pancreatic ribonuclease A, refined by a restrained parameter least squares procedure at 2.0 A resolution, and rebuilt using computer graphics. The final agreement factor (formula see text) is 0.159. The positions of the 951 main chain atoms have been determined with an estimated accuracy of 0.17 A. In addition, the model includes a phosphate group in the active site and 176 waters, many of them with partial occupancy. The bond lengths in the refined structure of RNase A differ from the ideal values by an overall root mean square deviation of 0.022 A; the corresponding value for angle distances is 0.06 A. The root mean square deviation of planar atoms from ideality is 0.017 A, and root mean square deviation of the peptide torsion angles from 180 degrees is 3.4 degrees. The model is in good agreement with the final difference Fourier maps. Two active site histidines, His 12 and His 119, form hydrogen bonds to the phosphate ion. His 119 is also hydrogen bonded to the carboxyl of ASp 121 and His 12 to the carbonyl of Thr 45. The structure of the RNase A is very similar to that of RNase S, particularly in the active site region. The root mean square discrepancy of all atoms from residues 1 to 16 and 24 to 123 is 1.06 A and the root mean square discrepancy for the active site region is 0.6 A.     

Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis

Characterization of patterns of ribosomal RNA (rRNA) homology with restriction digests of Bacillus subtilis 168 chromosomal DNA and with cloned DNA sequences has resulted in the construction of a physical map of the rRNA gene sets. There are two types of gene sets which differ in the size of "spacer" DNA sequences separating the 16S and 23S rRNA determinants. It was estimated that there are ten rRNA gene sets on the B. subtilis chromosome.     

Genomic organization, chromosomal localization, alternative splicing, and isoforms of the human synaptosome-associated protein-23 gene implicated in vesicle-membrane fusion processes

Synaptosome-associated protein-23 (SNAP23) is a component of the cellular mechanism required for specific membrane fusion and targetting of intracellular vesicles. We have cloned the full-length human cDNA and the SNAP23 gene. The SNAP23 gene has eight exons, with the initiation codon located in exon 2, and maps to the human chromosome 15q21-22 region. The human SNAP23 gene can generate two types of message, the full-length message (SNAP23A) and a shorter message (SNAP23B). The latter is the result of alternative splicing where exon 5 is joined to exon 7 and the skipping of exon 6; it thus lacks a region that is required for non-specific binding to plasma membranes. The two isoforms, expressed as fusion proteins with glutathione-S-transferase, interact in vitro with human syntaxin 6, thus retaining the specific protein interaction required for membrane fusion. Alterations in the SNAP23 gene might be involved in neurological and other diseases with defects in vesicle-membrane fusion processes that map to 15q15-21.     

Molecular Characteristics of Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae from Humans in the Community

Objective

To investigate the molecular characteristics of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae collected during a cross-sectional study examining the prevalence and risk factors for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in humans living in areas with high or low broiler density.

Methods

ESC-resistant Enterobacteriaceae were identified by combination disc-diffusion test. ESBL/AmpC/carbapenemase genes were analysed using PCR and sequencing. For E. coli, phylogenetic groups and MLST were determined. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing.

Results

175 ESC-resistant Enterobacteriaceae were cultured from 165/1,033 individuals. The isolates were Escherichia coli(n=65), Citrobacter freundii (n=52), Enterobacter cloacae (n=38), Morganella morganii (n=5), Enterobacter aerogenes (n=4), Klebsiella pneumoniae (n=3), Hafnia alvei (n=2), Shigella spp. (n=2), Citrobacter amalonaticus (n=1), Escherichia hermannii (n=1), Kluyvera cryocrescens (n=1), and Pantoea agglomerans (n=1). The following ESBL genes were recovered in 55 isolates originating from 49 of 1,033 (4.7 %) persons: bla _CTX-M-1 (n=17), bla _CTX-M-15 (n=16), bla _CTX-M-14 (n=9), bla _CTX-M-2 (n=3), bla _CTX-M-3 (n=2), bla _CTX-M-24 (n=2), bla _CTX-M-27 (n=1), bla _CTX-M-32 (n=1), bla _SHV-12 (n=2), bla _SHV-65 (n=1) and bla _TEM-52 (n=1). Plasmidic AmpC (pAmpC) genes were discovered in 6 out of 1,033 (0.6 %) persons. One person carried two different E. coli isolates, one with bla _CTX-M-1 and the other with bla _CMY-2 and therefore the prevalence of persons carrying Enterobacteriaceae harboring ESBL and/or pAmpC genes was 5.2 %. In eight E. coli isolates the AmpC phenotype was caused by mutations in the AmpC promoter region. No carbapenemase genes were identified. A large variety of E. coli genotypes was found, ST131 and ST10 being most common.

Conclusions

ESBL/pAmpC genes resembled those from patients in Dutch hospitals, indicating that healthy humans form a reservoir for transmission of these determinants to vulnerable people. The role of poultry in the transmission to humans in the community remains to be elucidated.     

Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis

In a manner similar to ubiquitin, the prokaryotic ubiquitin‐like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome‐lacking non‐pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni²⁺‐chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI‐TOF‐MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.     

Ocean acidification reduces induction of coral settlement by crustose coralline algae

Crustose coralline algae (CCA) are a critical component of coral reefs as they accrete carbonate for reef structure and act as settlement substrata for many invertebrates including corals. CCA host a diversity of microorganisms that can also play a role in coral settlement and metamorphosis processes. Although the sensitivity of CCA to ocean acidification (OA) is well established, the response of their associated microbial communities to reduced pH and increased CO₂ was previously not known. Here we investigate the sensitivity of CCA‐associated microbial biofilms to OA and determine whether or not OA adversely affects the ability of CCA to induce coral larval metamorphosis. We experimentally exposed the CCA Hydrolithon onkodes to four pH/pCO₂ conditions consistent with current IPCC predictions for the next few centuries (pH: 8.1, 7.9, 7.7, 7.5, pCO₂: 464, 822, 1187, 1638 μatm). Settlement and metamorphosis of coral larvae was reduced on CCA pre‐exposed to pH 7.7 (pCO₂ = 1187 μatm) and below over a 6‐week period. Additional experiments demonstrated that low pH treatments did not directly affect the ability of larvae to settle, but instead most likely altered the biochemistry of the CCA or its microbial associates. Detailed microbial community analysis of the CCA revealed diverse bacterial assemblages that altered significantly between pH 8.1 (pCO₂ = 464 μatm) and pH 7.9 (pCO₂ = 822 μatm) with this trend continuing at lower pH/higher pCO₂ treatments. The shift in microbial community composition primarily comprised changes in the abundance of the dominant microbes between the different pH treatments and the appearance of new (but rare) microbes at pH 7.5. Microbial shifts and the concomitant reduced ability of CCA to induce coral settlement under OA conditions projected to occur by 2100 is a significant concern for the development, maintenance and recovery of reefs globally.