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Archives of Microbiology (v.186, #2)

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia by Michał Kalita; Tomasz Stępkowski; Barbara Łotocka; Wanda Małek (pp. 87-97).

Pairwise comparisons of Genista tinctoria (dyer’s weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer’s weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.

Keywords: Phylogeny of symbiotic genes; Genista tinctoria bradyrhizobia; Nodule structure; Symbiotic properties

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans by Markus John; Roland P. H. Schmitz; Martin Westermann; Walter Richter; Gabriele Diekert (pp. 99-106).

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.

Keywords: Dehalorespiration; Sulfurospirillum multivorans ; PceA; Tetrachloroethene reductive dehalogenase; Enzyme localization; Freeze fracture replica immunogold labeling

Iron nutrition and physiological responses to iron stress in Nitrosomonas europaea by Xueming Wei; Neeraja Vajrala; Loren Hauser; Luis A. Sayavedra-Soto; Daniel J. Arp (pp. 107-118).

Nitrosomonas europaea, as an ammonia-oxidizing bacterium, has a high Fe requirement and has 90 genes dedicated to Fe acquisition. Under Fe-limiting conditions (0.2 μM Fe), N. europaea was able to assimilate up to 70% of the available Fe in the medium even though it is unable to produce siderophores. Addition of exogenous siderophores to Fe-limited medium increased growth (final cell mass). Fe-limited cells had lower heme and cellular Fe contents, reduced membrane layers, and lower NH₃- and NH₂OH-dependent O₂ consumption activities than Fe-replete cells. Fe acquisition-related proteins, such as a number of TonB-dependent Fe-siderophore receptors for ferrichrome and enterobactin and diffusion protein OmpC, were expressed to higher levels under Fe limitation, providing biochemical evidence for adaptation of N. europaea to Fe-limited conditions.

Keywords: Nitrosomonas europaea ; Iron metabolism; Iron stress; Siderophore receptors

Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots by Juan Ignacio Quelas; Silvina L. López-García; Adriana Casabuono; M. Julia Althabegoiti; Elías J. Mongiardini; Julieta Pérez-Giménez; Alicia Couto; Aníbal R. Lodeiro (pp. 119-128).

The exopolysaccharide (EPS) is an extracellular molecule that in Bradyrhizobium japonicum affects bacterial efficiency to nodulate soybean. Culture conditions such as N availability, type of C-source, or culture age can modify the amount and composition of EPS. To better understand the relationship among these conditions for EPS production, we analyzed their influence on EPS in B. japonicum USDA 110 and its derived mutant ΔP22. This mutant has a deletion including the 3′ region of exoP, exoT, and the 5′ region of exoB, and produces a shorter EPS devoid of galactose. The studies were carried out in minimal media with the N-source at starving or sufficient levels, and mannitol or malate as the only C-source. Under N-starvation there was a net EPS accumulation, the levels being similar in the wild type and the mutant with malate as the C-source. By contrast, the amount of EPS diminished in N-sufficient conditions, being poyhydroxybutyrate accumulated with culture age. Hexoses composition was the same in both N-situations, either with mannitol or malate as the only C-source, in contrast to previous observations made with different strains. This result suggests that the change in EPS composition in response to the environment is not general in B. japonicum. The wild type EPS composition was 1 glucose:0.5 galactose:0.5 galacturonic acid:0.17 mannose. In ΔP22 the EPS had no galactose but had galacturonic acid, thus indicating that it was not produced from oxidation of UDP-galactose. Infectivity was lower in ΔP22 than in USDA 110. When the mutant infectivity was compared between N-starved or N-sufficient cultures, the N-starved were not less infective, despite the fact that the amounts of altered EPS produced by this mutant under N-starvation were higher than in N-sufficiency. Since this altered EPS does not bind soybean lectin, the interaction of EPS with this protein was not involved in increasing ΔP22 infectivity under N-starvation.

Keywords: Bradyrhizobium japonicum ; EPS; Nitrogen; Infectivity; Symbiosis; Soybean

Novel thermostable single-stranded DNA-binding protein (SSB) from Deinococcus geothermalis by Paweł Filipkowski; Anna Duraj-Thatte; Józef Kur (pp. 129-137).

To study the biochemical properties of single-stranded DNA-binding (SSB) protein from Deinococcus geothermalis (DgeSSB), we have cloned the ssb gene obtained by PCR and developed an overexpression system. The gene consists of an open reading frame of 900 nucleotides encoding a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa. The amino acid sequence exhibits 43, 44 and 75% identity with Thermus aquaticus, Thermus thermophilus and Deinococcus radiodurans SSBs, respectively. We show that DgeSSB is similar to Thermus/Deinococcus SSB in its biochemical properties. DgeSSB includes two oligonucleotide/oligosaccharide-binding folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DgeSSB bound about 30 nt independent of the salt concentration, and the fluorescence was quenched by about 65%. In a complementation assay in Escherichia coli, DgeSSB took over the in vivo function of EcoSSB. DgeSSB is thermostable with half-lives of 50 min at 70°C and 5 min at 90°C. Hence, DgeSSB offers an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and for analytical purposes.

Keywords: Deoxyribonucleic acid (DNA) replication; Expression; Purification; Thermophilic bacteria; Thermostability

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures by Xiaofeng Guo; Mary E. Lidstrom (pp. 139-149).

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-β-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C₁ metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.

Keywords: Methylobacterium extorquens AM1; Methylotroph; Physiology; Nucleotides; PHB; Enzyme activities

Genomic comparison of Yersinia pestis and Yersinia pseudotuberculosis by combination of suppression subtractive hybridization and DNA microarray by Xiaoyi Wang; Dongsheng Zhou; Long Qin; Erhei Dai; Jianguo Zhang; Yanping Han; Zhaobiao Guo; Yajun Song; Zongmin Du; Jin Wang; Jun Wang; Ruifu Yang (pp. 151-159).

In order to further figure out the genetic differences between Yersinia pestis and Yersinia pseudotuberculosis, and to provide novel insights into the evolution of Y. pestis, we compared the genomes of Y. pseudotuberculosis serogroup I strain ATCC29833 and Y. pestis Antiqua strain 49006 using a combination of suppression subtractive hybridization (SSH) and comparative genomic hybridization with DNAs from a diverse panel of Y. pestis and Y. pseudotuberculosis strains. SSH followed by BLAST analysis revealed 112 SSH fragments specific to strain ATCC29833, compared to the genomic sequence data of Y. pestis strains CO92, KIM and 91001. We identified 17 SSH fragments that appeared to be newly determined genetic contents of Y. pseudotuberculosis. The combination of SSH and microarray analysis showed that the parallel loss of genes contributed greatly not only to the significant genomic divergence between Y. pestis and Y. pseudotuberculosis but also to the intra-species microevolution of both of species. The results confirmed our earlier hypothesis that Y. pestis Antiqua isolates from the natural plague focus B in China represented the most ancestral strains in China, hence phylogenetically the closest isolates to Y. pseudotuberculosis.

Keywords: Y . pestis ; Y. pseudotuberculosis ; Suppression subtractive hybridization; DNA microarray; Genomic comparison

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Archives of Microbiology (v.186, #2)