Cytochrome cd1 Nitrite Reductase NirS Is Involved in Anaerobic Magnetite Biomineralization in Magnetospirillum gryphiswaldense and Requires NirN for Proper d1 Heme Assembly

The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd₁-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d₁ heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d₁ heme for holo-cytochrome cd₁ assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.     

The role of biogeography in shaping diversity of the intestinal microbiota in house mice

The microbial communities inhabiting the mammalian intestinal tract play an important role in diverse aspects of host biology. However, little is known regarding the forces shaping variation in these communities and their influence on host fitness. To shed light on the contributions of host genetics, transmission and geography to diversity in microbial communities between individuals, we performed a survey of intestinal microbial communities in a panel of 121 house mice derived from eight locations across Western Europe using pyrosequencing of the bacterial 16S rRNA gene. The host factors studied included population structure estimated by microsatellite loci and mitochondrial DNA, genetic distance and geography. To determine whether host tissue (mucosa)‐associated communities display properties distinct from those of the lumen, both the caecal mucosa and contents were examined. We identified Bacteroides, Robinsoniella and Helicobacter as the most abundant genera in both the caecal content and mucosa‐associated communities of wild house mice. Overall, we found geography to be the most significant factor explaining patterns of diversity in the intestinal microbiota, with a comparatively weaker influence of host population structure and genetic distance. Furthermore, the influence of host genetic distance was limited to the mucosa communities, consistent with this environment being more intimately coupled to the host.     

Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA

Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.     

A high risk phenotype of hypertrophic cardiomyopathy associated with a compound genotype of two mutated β-myosin heavy chain genes

Hypertrophic cardiomyopathy (HCM) is a genetically and clinically heterogeneous myocardial disease that is in most cases familial and transmitted in a dominant fashion. The most frequently affected gene codes for the cardiac (ventricular) β-myosin heavy chain. We have investigated the genetic cause of an isolated case of HCM, which was marked by an extremely severe phenotype and a very early age of onset. HCM is normally not a disease of small children. The proband was a boy who had suffered cardiac arrest at the age of 6.5years (resuscitation by cardioconversion). Upon screening of the β-myosin heavy chain gene as a candidate, two missense mutations, one in exon19 (Arg719Trp) and a second in exon12 (Met349Thr), were identified. The Arg719Trp mutation was de novo, as it was not found in the parents. In contrast, the Met349Thr mutation was inherited through the maternal grandmother. Six family members were carriers of this mutation but only the proband was clinically affected. Segregation and molecular analysis allowed us to assign the Met349Thr mutation to the maternal and the Arg719Trp de novo mutation to the paternal β-myosin allele. Thus, the patient has no normal myosin. We interpret these findings in terms of compound heterozygosity of a dominant (Arg719Trp) and a recessive (Met349Thr) mutation. Whereas a single mutated Arg719Trp allele would be sufficient to cause HCM, the concurrent Met349Thr mutation alone does not apparently induce the disease. Nevertheless, it conceivably contributes to the particularly severe phenotype. Received: 15 September 1997 / Accepted: 26 November 1997     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Using body mass dynamics to examine long-term habitat shifts of arctic-molting geese: evidence for ecological change

From 1976 onward, molting brant geese (Branta bernicla) within the Teshekpuk Lake Special Area, Alaska, shifted from inland, freshwater lakes toward coastal wetlands. Two hypotheses explained this redistribution: (1) ecological change: redistribution of molting brant reflects improvements in coastal foraging habitats, which have undergone a succession toward salt-tolerant plants due to increased coastal erosion and saltwater intrusion as induced by climate change or (2) interspecific competition: greater white-fronted geese (Anser albifrons) populations increased 12-fold at inland lakes, limiting food availability and forcing brant into coastal habitats. Both hypotheses presume that brant redistributions were driven by food availability; thus, body mass dynamics may provide insight into the relevance of these hypotheses. We compared body mass dynamics of molting brant across decades (1978, 1987–1992, 2005–2007) and, during 2005–2007, across habitats (coastal vs. inland). Brant lost body mass during molt in all three decades. At inland habitats, rates of mass loss progressively decreased by decade despite the increased number of greater white-fronted geese. These results do not support an interspecific competition hypothesis, instead suggesting that ecological change enhanced foraging habitats for brant. During 2005–2007, rates of mass loss did not vary by habitat. Thus, while habitats have improved from earlier decades, our results cannot distinguish between ecological changes at inland versus coastal habitats. However, we speculate that coastal forage quality has improved beyond that of inland habitats and that the body mass benefits of these higher quality foods are offset by the disproportionate number of brant now molting coastally.     

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-gamma-deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.