Evidence For Ahl Autoinducer Production By The Soybean Symbiont Brad Yrhizobium Japonicum

D.J. Westenberg

Department of Biological Sciences, University of Missouri - Rolla, MO, USA

During the rhizobium/legume symbiosis, the bacterial partner must make the transition from a free-living organism to an intracellular symbiont (bacteroid) capable of nitrogen fixation. Many bacterial genes are regulated in response to the transition from free-living bacterium to bacteroid. For example, attachment proteins would no longer be needed but nitrogenase would be needed. In addition, it would not be wise for the nitrogen-fixing bacteroid to begin fixing nitrogen until a sufficient cell density is achieved.

A number of symbiotic and pathogenic bacteria regulate the expression of symbiosis or virulence specific genes in response to cell density (quorum sensing). In a quorum sensing regulatory system, the bacterium produces an autoinducer molecule (AI) that is secreted to the surrounding medium. Once the AI reaches a high concentration, the AI interacts with regulatory proteins that either activate or repress specific genes. In gram-negative bacteria, two types of AIs have been observed (AI-1 and AI-2). AI-1 molecules are A'-acyl-homoserine lactones (AHL) and the structure of AI-2 has not yet been determined. Recently, evidence for a peptide AI molecule in B. japonicum has been presented. AHL AIs have been detected in Rhizobium leguminosarum and Rhizobium meliloti. However, to date, AHL autoinducers have not been detected in the soybean symbiont, B. japonicum.

Using the NTL4/pZLR4 indicator strain described by Piper et al., we screened twelve strains of B. japonicum. Three of the twelve strains (61A1186, 61A224, and 61A227) produce AHLs (Figure 1). To our knowledge, this is the first evidence of

AHL autoinducer production in B. japonicum. The number and type of autoinducer molecule(s) is currently being pursued along with experiments to measure the time course of autoinducer production and optimization of autoinducer production. Autoinducer production by B. japonicum is a potential target for improving the competitiveness of inoculum strains and we are using the indicator strain to screen gene libraries for the genes responsible for autoinducer production and the genes that may be regulated by these molecules.

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Figure 1. Induction of lacZ expression by NTL4/pZLR4 in response to culture supernatants from various B. japonicum and control strains.

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HEME-DEPENDENT DEGRADATION OF THE Irr PROTEIN FROM BRAD YRHIZOBIUM JAPONICUM

Department of Biochemistry, State University of New York at Buffalo, NY 14214, USA

1. Introduction

The bacterium, Bradyrhizobium japonicum, can establish a symbiotic relationship with soybean plants that is manifested as root nodules. Heme is essential for respiratory metabolism and nitrogen fixation and, therefore, we are interested in the regulation of heme biosynthetic pathway. In B japonicum, the heme biosynthetic pathway is regulated by iron via the iron response regulator (Irr) protein. Under iron limitation, Irr accumulates and negatively regulates hemB, the gene encoding the heme biosynthesis enzyme ALA dehydratase. However, when iron is sufficient, Irr degrades to derepress the pathway (Hamza et al. 1998). This degradation is mediated by heme, which binds directly to the protein at the heme regulatory motif (HRM) (Qi et al. 1999). Here, we address the mechanism of heme-mediated Irr degradation in vitro.

2. Results and Discussion

Purified Irr was degraded by hemin in the presence of oxygen and a reducing agent (DTT). The redox inactive heme analog zinc protoporphyrin and free ferric iron did not catalyze degradation. In addition, Irr was oxidatively carbonylated during the degradation process. These data indicate that degradation of Irr involves oxidative carbonylation of the protein. Carbonylated products both smaller and larger than the Irr monomer were observed, suggesting both oxidative cleavage and peptide cross-linking, respectively. The Irr mutant IrrC29A does not bind heme with high affinity, and is stable in vivo in the presence of iron (Qi et al. 1999). IrrC29A was carbonylated to a much lesser extent than the wild type protein. Furthermore, oxygen was required for iron-dependent degradation in vivo. We suggest that heme binding to Irr at the HRM catalyzes the localized generation of reactive oxygen species leading to Irr oxidation and degradation.

3. References

Hamza I et al. (1998) J. Biol. Chem. 273, 21669-21674 Qi Z et al. (1999) Proc. Natl. Acad. Sci. USA 96,13056-13061

FUNCTIONAL CHARACTERIZATION OF THREE Pn HOMOLOGS IN RHODOSPIRILL UM R UBR UM

Y. Zhang1'2'3, E.L. Pohlmannu,P.W. Ludden2'3, G.P. Roberts1'3

'Department of Bacteriology department of Biochemistry

3The Center for the Study of Nitrogen Fixation, University of Wisconsin - Madison, Madison, WI53706, USA

The GlnB (Pn) protein, the gene product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. We recently have identified two other Pn homologs in that organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, they have both distinct and overlapping functions in the cell. While GlnB is required for the activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in that process. In contrast, either GlnB or GlnJ can serve as a critical element in the regulation of the reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the DRAT/DRAG regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of these proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of DRAT/DRAG system, GlnB and GlnJ appear to be responsive to not only changes in nitrogen status, but also changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

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