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Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO(2) to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an alpha-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active alpha-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and delta(13)C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH(3) protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule delta(13)C abundance suggest the recycling of at least part of the HCO(3)(-) produced by CAA1.

Protection against reactive oxygen species (ROS) is important for legume-nodulating rhizobia during the establishment and maintenance of symbiosis, as well as under free-living conditions, because legume hosts might assail incoming microbes with ROS and because nitrogenase is extremely sensitive to ROS. We generated mutants of two potential catalase genes in Mesorhizobium loti MAFF303099 to investigate their physiological significance. Biochemical results indicated that genes with the locus tags mlr2101 and mlr6940 encoded a monofunctional catalase and a bifunctional catalase-peroxidase, respectively, that were named katE and katG. Under free-living conditions, the katG mutant demonstrated an extended generation time and elevated sensitivity to exogenous H(2)O(2), whereas the katE mutant exhibited no generation time extension and only a slight increase in sensitivity to exogenous H(2)O(2). However, the katE mutant showed a marked decrease in its survival rate during the stationary phase. With regard to symbiotic capacities with Lotus japonicus, the katG mutant was indistinguishable from the wild type; nevertheless, the mutants with disrupted katE formed nodules with decreased nitrogen fixation capacities (about 50 to 60%) compared to those formed by the wild type. These mutant phenotypes agreed with the expression profiles showing that transcription of katG, but not katE, was high during the exponential growth phase and that transcription levels of katE versus sigA were elevated during stationary phase and were approximately fourfold higher in bacteroids than mid-exponential-phase cells. Our results revealed functional separation of the two catalases, as well as the importance of KatE under conditions of strong growth limitation.

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.

The first step in the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum has been believed for more than a decade to be conversion of PCP to tetrachlorohydroquinone. We show here that PCP is actually converted to tetrachlorobenzoquinone, which is subsequently reduced to tetrachlorohydroquinone by PcpD, a protein that had previously been suggested to be a PCP hydroxylase reductase. pcpD is immediately downstream of pcpB, the gene encoding PCP hydroxylase (PCP monooxygenase). Expression of PcpD is induced in the presence of PCP. A mutant strain lacking functional PcpD has an impaired ability to remove PCP from the medium. In contrast, the mutant strain removes tetrachlorophenol from the medium at the same rate as does the wild-type strain. These data suggest that PcpD catalyzes a step necessary for degradation of PCP, but not for degradation of tetrachlorophenol. Based upon the known mechanisms of flavin monooxygenases such as PCP hydroxylase, hydroxylation of PCP should produce tetrachlorobenzoquinone, while hydroxylation of tetrachlorophenol should produce tetrachlorohydroquinone. Thus, we proposed and verified experimentally that PcpD is a tetrachlorobenzoquinone reductase that catalyzes the NADPH-dependent reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone.

The reproduction and morphogenesis of Rickettsiella chironomi, an unusual procaryotic parasite of midge larvae, was studied with electron microscopy. The morphogenic cycle was similar to that found in chlamydia and consisted of four major cell types: (i) medium-sized spherical initial bodies (ca. 1 micrometers in diameter), (ii) large spherical initial bodies (1.5-2 micrometers), (iii) spherical intermediate bodies (600 to 700 nm), and (iv) disk-shaped elementary bodies (60 X 600 nm). The primary mode of reproduction involved binary fission of initial bodies to form other initial bodies or intermediate bodies. Each intermediate body condensed, forming an elementary body. The morphogenesis of R. chironomi is compared with that of several other organisms to which it is possibly related, including vertebrate chlamydia and invertebrate pathogens of the genera Rickettsiella and Porochlamydia, and its taxonomic position in regard to these is discussed. Additionally, a brief description of the pathology caused by the development of R. chironomi in larvae of Chironomus decorus and Chironomus frommeri is given.