ABSTRACTBacterial Lon proteases play important roles in a variety of biological processes in addition to housekeeping functions. In this study, we focused on the Lon protease ofAzorhizobium caulinodans, which can fix nitrogen both during free-living growth and in stem nodules of the legumeSesbania rostrata. The nitrogen fixation activity of anA. caulinodanslonmutant in the free-living state was not significantly different from that of the wild-type strain. However, the stem nodules formed by thelonmutant showed little or no nitrogen fixation activity. By microscopic analyses, two kinds of host cells were observed in the stem nodules formed by thelonmutant. One type has shrunken host cells containing a high density of bacteria, and the other type has oval or elongated host cells containing a low density or no bacteria. This phenotype is similar to apraRmutant highly expressing therebgenes. Quantitative reverse transcription-PCR analyses revealed thatrebgenes were also highly expressed in thelonmutant. Furthermore, alon rebdouble mutant formed stem nodules showing higher nitrogen fixation activity than thelonmutant, and shrunken host cells were not observed in these stem nodules. These results suggest that Lon protease is required to suppress the expression of therebgenes and that high expression ofrebgenes in part causes aberrance in theA. caulinodans-S. rostratasymbiosis. In addition to the suppression ofrebgenes, it was found that Lon protease was involved in the regulation of exopolysaccharide production and autoagglutination of bacterial cells.

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G2/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10.

No abstract

HIV-1 budding requires interaction between Gag and cellular TSG101 to initiate viral particle assembly and release via the endosomal sorting complexes required for transport (ESCRT) pathway. However, some reports show that overexpression of TSG101 inhibits virus release by disruption of Gag targeting process. Since a HIV-1 accessory protein, Vpr binds to Gag p6 domain at the position close to the binding site for TSG101, whether Vpr implicates TSG101 overexpression effect has not been investigated. Here, we found that Vpr abrogates TSG101 overexpression effect to rescue viral production. Co-transfection of TSG101 and Gag with Vpr prevented TSG101-induced Gag accumulation in endosomes and lysosomes. In addition, Vpr rescued virus-like particle (VLP) production in a similar manner as a lysosomal inhibitor, Bafilomycin A1 indicating that Vpr inhibits TSG101-induced Gag downregulation via lysosomal pathway. Vpr and Gag interaction is required to counteract TSG101 overexpression effect since Vpr A30F mutant which is unable to interact with Gag and incorporate into virions, reduced ability to prevent Gag accumulation and to rescue VLP production. In addition, GST pull-down assays and Biacore analysis revealed that Vpr competed with TSG101 for Gag binding. These results indicate that Vpr overcomes the effects of TSG101 overexpression to support viral production by competing with TSG101 to bind Gag.

Azorhizobium caulinodans is a microsymbiont of Sesbania rostrata Bremek. & Oberm., and is able to fix nitrogen in both the free-living and symbiotic states. In this study, we focused on the ggm gene (locus tag, AZC_4606) that encodes a putative membrane protein belonging to the TIGR02302 family. Although the genes encoding TIGR02302 family protein are distributed in a wide range of alphaproteobacteria including rhizobia, the functions of this protein are still unknown. To investigate the functions of this protein in A. caulinodans, we made a ggm mutant, and analyzed its phenotypes. The ggm mutant produced more bubbles than the wild-type strain in L3 + N medium liquid cultures, and formed mucoid colonies on L3 + N medium agar plates, suggesting that the ggm mutant overproduced exopolysaccharides (EPSs). The amounts of EPSs produced by the ggm mutant on L3 + N plates were about 1.3-fold higher than those by the wild-type strain, and expression levels of EPS productionrelated genes in the ggm mutant grown in L3 + N liquid medium were about 2-to 4-fold higher than those of the wild-type strain. In addition, the stem nodules formed by the ggm mutant on the stems of S. rostrata showed little or no nitrogen-fixing activity. By microscopic analyses, large infection pockets and a few infected cells were observed in the stem nodules formed by ggm mutant, suggesting that the ggm mutant is defective in invasion into plant cells. Taken together, our results suggest that Ggm is involved in EPS production and that adequate levels of EPS production are required for A. caulinodans to invade into host cells.

2005). LPS is composed of three parts: lipid A, containing sugar and fatty acid, which forms the outer leaflet of the outer membrane and anchors LPS to the cell envelope; a core oligosaccharide region, a non-repeating oligosaccharide, which links lipid A and O-antigen; and the Oantigen, consisting of repeating oligosaccharide units. The LPS containing or lacking O-antigen is usually called smooth LPS and rough LPS, respectively. In the majority of gram-negative bacteria, the core oligosaccharide can be subdivided into an outer core and an inner core. The outer core region provides an attachment site for O-antigen. The inner core typically contains residues of 3-deoxy-D -manno-2-octulosonic acid (Kdo) and L-glycero-Dmanno-heptose. Kdo connects the inner core to the lipid A (Heinrichs et al., 1998;Raetz and Whitfield, 2002).Azorhizobium caulinodans ORS571 is a microsymbiont of a tropical legume, Sesbania rostrata (Dreyfus and Dommergues, 1981;Dreyfus et al., 1983Dreyfus et al., , 1988. N 2 -fixing nodules are formed by A. caulinodans on the stems as well as on the roots of S. rostrata. Previously, the whole genome sequence of A. caulinodans was determined (Lee et al., 2008), and we performed a concurrent large-scale screening of rhizobial genetic factors involved in nodule development using A. caulinodans mutants created by random Tn5 mutagenesis (Suzuki et al., 2007). As a result of this screening, we isolated three mutants having a Tn5-insertion in the putative LPS biosynthesis genes (Suzuki et al., 2007). These three mutants, Ao13-C11, Ao77-C09 and Ao80-F04, were disrupted in the rfaF, rfaD, and rfaE genes, respectively. These genes are involved in the synthesis of the LPS inner core region. The rfaD and rfaE genes encode ADP-L-glycero-D-manno-heptose-6-epimerase and ADP-L-glycero-D-manno-heptose synthase, respectively. These enzymes are involved in the synthesis of ADP-L-glycero-D-manno-heptose, which is a component of the LPS inner core (Kneidinger et al., 2002; The lipopolysaccharide (LPS) of Azorhizobium caulinodans ORS571, which forms N 2 -fixing nodules on the stems and roots of Sesbania rostrata, is known to be a positive signal required for the progression of nodule formation. In this study, four A. caulinodans mutants producing a variety of defective LPSs were compared. The LPSs of the mutants having Tn5 insertion in the rfaF, rfaD, and rfaE genes were more truncated than the modified LPSs of the oac2 mutants. However, the nodule formation by the rfaF, rfaD, and rfaE mutants was more advanced than that of the oac2 mutant, suggesting that invasion ability depends on the LPS structure. Our hypothesis is that not only the wild-type LPSs but also the altered LPSs of the oac2 mutant may be recognized as signal molecules by plants. The altered LPSs may act as negative signals that halt the symbiotic process, whereas the wild-type LPSs may prevent the halt of the symbiotic process. The more truncated LPSs of the rfaF, rfaD, and rfaE mutants perhaps no longer function as negative signals inducing discontinu...

Bacteria have multiple K ϩ uptake systems. Escherichia coli, for example, has three types of K ϩ uptake systems, which include the low-K ϩ -inducible KdpFABC system and two constitutive systems, Trk (TrkAG and TrkAH) and Kup. Azorhizobium caulinodans ORS571, a rhizobium that forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, also has three types of K ϩ uptake systems. Through phylogenetic analysis, we found that A. caulinodans has two genes homologous to trkG and trkH, designated trkI and trkJ. We also found that trkI is adjacent to trkA in the genome and these two genes are transcribed as an operon; however, trkJ is present at a distinct locus. Our results demonstrated that trkAI, trkJ, and kup were expressed in the wild-type stem nodules, whereas kdpFABC was not. Interestingly, Δkup and Δkup ΔkdpA mutants formed Fix -nodules, while the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant formed Fix ϩ nodules, suggesting that with the additional deletion of Trk system genes in the Δkup mutant, Fix ϩ nodule phenotypes were recovered. kdpFABC of the Δkup ΔtrkJ mutant was expressed in stem nodules, but not in the free-living state, under high-K ϩ conditions. However, kdpFABC of the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant was highly expressed even under high-K ϩ conditions. The cytoplasmic K ϩ levels in the Δkup ΔtrkA ΔtrkI mutant, which did not express kdp-FABC under high-K ϩ conditions, were markedly lower than those in the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant. Taking all these results into consideration, we propose that TrkJ is involved in the repression of kdpFABC in response to high external K ϩ concentrations and that the TrkAI system is unable to function in stem nodules.IMPORTANCE K ϩ is a major cytoplasmic cation in prokaryotic and eukaryotic cells. Bacteria have multiple K ϩ uptake systems to control the cytoplasmic K ϩ levels. In many bacteria, the K ϩ uptake system KdpFABC is expressed under low-K ϩ conditions. For years, many researchers have argued over how bacteria sense K ϩ concentrations. Although KdpD of Escherichia coli is known to sense both cytoplasmic and extracellular K ϩ concentrations, the detailed mechanism of K ϩ sensing is still unclear. In this study, we propose that the transmembrane TrkJ protein of Azorhizobium caulinodans acts as a sensor for the extracellular K ϩ concentration and that high extracellular K ϩ concentrations repress the expression of KdpFABC via TrkJ. KEYWORDS potassium transport, rhizobium, symbiosisA zorhizobium caulinodans ORS571 is a microsymbiont of the water-tolerant tropical legume Sesbania rostrata (1-3), where it forms N 2 -fixing nodules on the stems and roots. A previous transposon mutagenesis study on the rhizobial factors involved in

From the infection pockets, infection threads guide the bacteria toward the nodule meristematic zone, wherein the bacteria are released into the host cells and surrounded by a plant-derived peribacteroid membrane (Tsien et al., 1983). The infected host cells are filled with differentiated bacteroids, and the infected area enlarges as the nodule matures (Dreyfus and Dommergues, 1981; Tsien et al., 1983). A. caulinodans can potentially kill the host plant cells by producing R-bodies (Matsuoka et al., 2017b). R-bodies are large proteinaceous ribbons that are coiled into cylindrical structures and were first observed in Caedibacter species, which are obligate bacterial endosymbionts of paramecia (Anderson et al., 1964). The paramecia that harbor the R-body-producing Caedibacter cells (i.e., killer paramecia) release the bacterial cells through their cytopyge. The paramecia without the endosymbiont (i.e., sensitive paramecia) ingest the released bacteria and die. A. caulinodans has a reb operon (locus tags on the genome, AZC_3781 to AZC_3788), containing four reb genes (AZC_3781, AZC_3782, AZC_3783, and AZC_3786), which is associated with R-body production (Heruth et al., 1994; Matsuoka et al., 2017b). In addition, three unknown genes (AZC_3784, AZC_3785, and AZC_3787), and a transcription factor gene, rebR (AZC_3788) are also contained in the reb operon. RebR binds to the promoter region of the reb operon and acts as an activator for the reb operon expression (Matsuoka et al., 2017b). The reb operon expression is usually suppressed by Localization of the reb operon expression is inconsistent with that of the R-body production in the stem nodules formed by Azorhizobium caulinodans mutants having a deletion of praR