Modifikationen der Atmungskette in Corynebacterium glutamicum\textit{Corynebacterium glutamicum} und Rolle des Flavohämoproteins Hmp

$\textit{Corynebacterium glutamicum}$ possesses a branched aerobic respiratory chain with two terminal oxidases, cytochrome bd oxidase with a high oxygen affinity but a low H$^{+}$/O ratio, and the proton pumping cytochrome aa$_{3}$ oxidase with a low oxygen affinity but a high H$^{+}$/O ratio. The latter forms a supercomplex with the cytochrome bc1 complex. Under aerobic conditions these two braches build up an electrochemical proton gradient, which, in the process of oxidative phosphorylation, is used for ATP synthesis by F$_{1}$F$_{O}$-ATP synthase. Within this work the respiratory chain of $\textit{C. glutamicum}$ was modified and the influences of these modifications were studied. The cytochrome bc$_{1}$-aa${3}$ supercomplex is the major proton pump in $\textit{C. glutamicum}$. To study the influence of increased cellular levels of the supercomplex, expression of its structural genes was enhanced two-fold by exchange of the native promoters with the P$_{tuf}$ promoter of elongation factor Tu. Higher levels of cytochromes a, b and c were detectable in the recombinant strain and resting cells showed a 30% increased oxygen consumption rate. However, growth was comparable to the wild type. In a similar approach, the native promoter of the atpBEFHAGDC operon was exchanged by P$_{tuf}$. Surprisingly this resulted in a 50-60% decreased expression of the $\textit{atp}$ genes, indicating that the native atp promoter is stronger than the $\textit{tuf}$ promoter and might be suitable for enhancing gene expression in producer strains of $\textit{C. glutamicum}$. In order to improve energy conservation under microaerobic conditions, attempts were made to replace the $\textit{bd}$ oxidase by a cytochrome $\textit{cbb}_{3}$-type oxidase from $\textit{Bradyrhizobium japonicum}$, a proton-pumping heme-copper type enzyme with a very high oxygen affinity. However, as the synthesis of the accessory protein FixG proved to be toxic for $\textit{C. glutamicum}$, functional expression of the heterologous $\textit{cbb}_{3}$-type oxidase could not be achieved. $\textit{C. glutamicum}$ possesses a limited potential for anaerobic growth by nitrate respiration via the nitrate reductase NarGHJI. However, due to the lack of a nitrite reductase, nitrite accumulates and inhibits growth. In order to allow conversion of nitrite to ammonia and thus the use of nitrate as a nitrogen source, attempts were made to express a dissimilatory nitrite reductase from $\textit{Wolinella succinogenes}$ and an assimilatory nitrite reductase from Mycobacterium smegmatis in $\textit{C. glutamicum}$. Unfortunately, anaerobic nitrite reduction did not work with either of these enzymes. In another aspect of this work, the influence of nitrite on growth and global gene expression was analyzed. The gene most strongly upregulated by nitrite was found to be $\textit{hmp}$ encoding a flavohaemoprotein. A $\Delta \textit{hmp}$ mutant was more sensitive towards nitrite and the NO-donating agent sodium nitroprusside under aerobic conditions. Anaerobic growth by nitrate respiration was also severely impaired. Thus flavohaemoprotein Hmp of $\textit{C. glutamicum}$ is important for coping with nitrite-derived stress under aerobic and anaerobic conditions

Parental alarm calls suppress nestling vocalization.

Evolutionary models suggest that the cost of a signal can ensure its honesty. Empirical studies of nestling begging imply that predator attraction can impose such a cost. However, parents might reduce or abolish this cost by warning young of the presence of danger. We tested, in a controlled field playback experiment, whether alarm calls cause 5-, 8- and 11-day-old nestlings of the white-browed scrubwren, Sericornis frontalis, to suppress vocalization. In this species, nestlings vocalize when parents visit the nest ('begging') and when they are absent ('non-begging'), so we measured effects on both types of vocalization. Playback of parental alarm calls suppressed non-begging vocalization almost completely but only slightly reduced begging calls during a playback of parental feeding calls that followed. The reaction of nestlings was largely independent of age. Our results suggest two reasons why experiments ignoring the role of parents probably overestimate the real cost of nestling vocalizations. Parents can warn young from a distance about the presence of danger and so suppress non-begging vocalizations that might otherwise be overheard, and a parent's presence at the nest presumably indicates when it is safe to beg

From nestling calls to fledgling silence: adaptive timing of change in response to aerial alarm calls

Young birds and mammals are extremely vulnerable to predators and so should benefit from responding to parental alarm calls warning of danger. However, young often respond differently from adults. This difference may reflect: (i) an imperfect stage in the gradual development of adult behaviour or (ii) an adaptation to different vulnerability. Altricial birds provide an excellent model to test for adaptive changes with age in response to alarm calls, because fledglings are vulnerable to a different range of predators than nestlings. For example, a flying hawk is irrelevant to a nestling in a enclosed nest, but is dangerous to that individual once it has left the nest, so we predict that young develop a response to aerial alarm calls to coincide with fledging. Supporting our prediction, recently fledged white-browed scrubwrens, Sericornis frontalis, fell silent immediately after playback of their parents' aerial alarm call, whereas nestlings continued to calling despite hearing the playback. Young scrubwrens are therefore exquisitely adapted to the changing risks faced during development

Adaptive differences in response to two types of parental alarm call in altricial nestlings

Vertebrate alarm calls can contain information about the type of predator and the degree of danger, but young animals often respond to alarm calls differently from adults. The distinct behaviour of young may reflect an imperfect stage in the gradual development of the adult response, or a response adapted to specific risks faced by young. In this study, we tested whether nestling white-browed scrubwrens, Sericornis frontalis, responded to different alarm calls according to their specific risks of predation. As predators on the ground pose a danger to scrubwren nestlings, whereas flying predators do not, we predicted that they would respond to ground alarm calls but not to aerial alarm calls. In a field playback experiment, we tested the response of young to aerial and ground alarm calls, each presented in a shorter (less urgent) and longer (more urgent) form. We found that both 5- and 11-day-old nestlings responded to ground alarm calls, and did so more strongly to the more urgent playback. By contrast, the response to aerial alarm calls started to develop only towards the end of the nestling stage. Thus, scrubwren nestlings can distinguish between different types of alarm calls and react more strongly to calls warning of a predator posing greater danger, appropriate to the nestling stage of development. Furthermore, they use the length of ground alarm calls as an indicator of the degree of danger

Conversion of Corynebacterium glutamicum from an aerobic respiring to an aerobic fermenting bacterium by inactivation of the respiratory chain

AbstractIn this study a comparative analysis of three Corynebacterium glutamicum ATCC 13032 respiratory chain mutants lacking either the cytochrome bd branch (ΔcydAB), or the cytochrome bc1–aa3 branch (Δqcr), or both branches was performed. The lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of a functional cytochrome bc1–aa3 supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly increased maintenance coefficient under oxygen excess. These results show that the bc1–aa3 supercomplex is of major importance for aerobic respiration. For the first time, a C. glutamicum strain with a completely inactivated aerobic respiratory chain was obtained (ΔcydABΔqcr), named DOOR (devoid of oxygen respiration), which was able to grow aerobically in BHI (brain–heart infusion) glucose complex medium with a 70% reduced biomass yield compared to the wild type. Surprisingly, reasonable aerobic growth was also possible in glucose minimal medium after supplementation with peptone. Under these conditions, the DOOR strain displayed a fermentative type of catabolism with l-lactate as major and acetate and succinate as minor products. The DOOR strain had about 2% of the oxygen consumption rate of the wild type, showing the absence of additional terminal oxidases. The pmf of the DOOR mutant was reduced by about 30% compared to the wild type. Candidates for pmf generation in the DOOR strain are succinate:menaquinone oxidoreductase, which probably can generate pmf in the direction of fumarate reduction, and F1FO-ATP synthase, which can couple ATP hydrolysis to the export of protons