Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea.

Magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats. Because of their fastidious requirements for growth conditions, only very few axenic MTB cultures have been obtained worldwide. In this study, we report a novel marine magnetotactic spirillum axenic culture, designated as QH-2, isolated from the China Sea. It was able to grow in semi-solid or liquid chemically defined medium. The cells were amphitrichously flagellated and contained one single magnetosome chain with an average number of 16 magnetosomes per cell. Phosphate and lipid granules were also observed in the cells. Both rock magnetism and energy-dispersive X-ray spectroscopy characterizations indicated that the magnetosomes in QH-2 were single-domain magnetites (Fe(3)O(4)). QH-2 cells swam mostly in a straight line at a velocity of 20-50 microm/s and occasionally changed to a helical motion. Unlike other magnetotactic spirilla, QH-2 cells responded to light illumination. As a consequence of illumination, the cells changed the direction in which they swam from parallel to the magnetic field to antiparallel. This response appears to be similar to the effect of an increase in [O(2)]. Analysis of the QH-2 16S rRNA sequence showed that it had greater than 11% sequence divergence from freshwater magnetotactic spirilla. Thus, the marine QH-2 strain seems to be both phylogenetically and magnetotactically distinct from the freshwater Magnetospirillum spp. studied previously.

Development and application of a bioluminescence-based test for assimilable organic carbon in reclaimed waters.

Assimilable organic carbon (AOC) is an important parameter governing the growth of heterotrophic bacteria in drinking water. Despite the recognition that variations in treatment practices (e.g., disinfection, coagulation, selection of filter media, and watershed protection) can have dramatic impacts on AOC levels in drinking water, few water utilities routinely measure AOC levels because of the difficulty of the method. To simplify the method, the Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX test bacteria were mutagenized by using luxCDABE operon fusion and inducible transposons to produce bioluminescent strains. The growth of these strains can easily be monitored with a programmable luminometer to determine the maximum cell yield via luminescence readings, and these values can be fitted to the classical Monod growth curve to determine bacterial growth kinetics and the maximum growth rate. Standard curves using acetate carbon (at concentrations ranging from 0 to 1,000 microg/liter) resulted in coefficients of determination (r(2)) between luminescence units and acetate carbon levels of 0.95 for P-17 and 0.89 for NOX. The bioluminescence test was used to monitor reclaimed water, in which average AOC levels range between 150 and 1,400 microg/liter acetate carbon equivalents. Comparison of the conventional AOC assay and the bioluminescent assay produced an r(2) of 0.92.

Proposal of Spirillum winogradskyi sp. nov., a novel microaerophilic species, an emended description of the genus Spirillum and Request for an Opinion regarding the status of the species Spirillum volutans Ehrenberg 1832.

A novel obligately organotrophic, facultatively microaerophilic spirillum, designated strain D-427(T), was isolated from sulfidic sludge of a municipal wastewater-treatment plant. Cells were Gram-negative, large and highly motile due to bipolar tufts of flagella covered with mucous sheaths. Coccoid cells were sometimes formed. Strain D-427(T) grew optimally at pH 7.5-7.8 and 28 degrees C in the presence of 2 % O(2) in the gas phase. The organism showed oxidase and very low catalase activity. The isolate grew chemo-organotrophically with a limited number of organic acids as substrates. The DNA G+C content was 38.0 mol% (T(m)). Phylogenetic analysis of the 16S rRNA gene sequence placed strain D-427(T) in the genus Spirillum within the class Betaproteobacteria. The 16S rRNA gene sequence similarity between strain D-427(T) and Spirillum volutans ATCC 19554(T), the type strain of the single species of the genus, was 98.6 %. The low level of DNA-DNA hybridization and different phenotypic properties indicate that strain D-427(T) is clearly distinguishable from Spirillum volutans. No strain of S. volutans is available from any established culture collection or from the authors who described this species. Therefore, on the basis of phenotypic and genotypic data and the fact that the type and single species of the genus Spirillum cannot be included in any scientific study, since the type strain has been lost, we propose to assign strain D-427(T) as a novel species of the genus Spirillum, Spirillum winogradskyi sp. nov. (type strain D-427(T) =DSM 12756(T) =VKM B-2518(T)), and we request that the Judicial Commission place the name Spirillum volutans on the list of rejected names if a suitable type strain is not found or a neotype is not proposed within 2 years following the publication of this paper. An emended description of the genus Spirillum is also provided.

Bacterial diversity in the bacterioneuston (sea surface microlayer): the bacterioneuston through the looking glass.

The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of approximately 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level.

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria.

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.

Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences.

Phylogenetic analysis of 15 species of the genus Aquaspirillum based on 16S rRNA gene (rDNA) sequences indicated that the genus Aquaspirillum is phylogenetically heterogeneous and the species could be divided into four groups as follows: Aquaspirillum serpens, the type species of this genus, A. dispar and A. putridiconchylium are situated in the family Neisseriaceae; members of the second group, A. gracile, A. delicatum, A. anulus, A. giesbergeri, A. sinuosum, A. metamorphum and A. psychrophilum, are included in the family Comamonadaceae; the two members of the third group, A. arcticum and A. autotrophicum, are included in the family Oxalobacteriaceae; and members of the fourth group, A. polymorphum, A. peregrinum, and A. itersonii, are included in the alpha-subdivision of Proteobacteria. Thus, phylogenetic studies indicated that all the species excepting A. serpens, the type species, should be transferred to distinct genera.

Identification of a gene for a rubrerythrin/nigerythrin-like protein in Spirillum volutans by using amino acid sequence data from mass spectrometry and NH2-terminal sequencing.

A hydrogen peroxide-resistant mutant of the catalase-negative microaerophile, Spirillum volutans, constitutively expresses a 21.5 kDa protein that is undetectable and non-inducible in the wild-type cells. Part of the gene that encodes the protein was cloned using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing. The deduced 158 amino acid polypeptide shows high relatedness to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris. The protein also shows high similarity to putative rubrerythrin proteins found in the anaerobic archeons Archaeoglobus fulgidus, Methanococcus jannaschii and Methanobacterium thermoautotrophicum. This is the first report of this type of protein in an organism that must respire with oxygen. It seems likely that the novel combination of methodologies used in this study could be applied to the rapid cloning of other genes in bacteria for which no genomic library yet exists.

Survival potential of Aeromonas hydrophila in freshwaters and nutrient-poor waters in comparison with other bacteria.

The survival of a genetically-marked Aeromonas hydrophila strain was studied in water microcosms using viable counts. Aeromonas hydrophila AWWX1 was shown to survive without decline in viable counts for at least 10 d in three of four filtered-autoclaved freshwaters (surface water and groundwater) and in all examined filtered-autoclaved nutrient-poor waters (bottled spring water, Milli-Q and tap water). However, in the unfiltered waters, a rapid decrease in viable counts of Aer. hydrophila AWWX1 was observed after 1-5 d. The survival of Aer. hydrophila AWWX1 in nutrient-poor waters was compared with that of Pseudomonas fluorescens P17 and Spirillum strain NOX. Survival characteristics were organism- and water-dependent. In the filtered-autoclaved waters, viable counts of Spirillum strain NOX were ca 1 log-unit higher than for Aer. hydrophila AWWX1 and Ps. fluorescens P17. The tested strains Aer. hydrophila AWWX1 and Ps. fluorescens P17 survived 3 to 20, respectively 2 to 4 times better in the filtered-autoclaved waters compared to the unfiltered waters. Apparently, any inherent capability of these micro-organisms to adapt to low-nutrient environments was undone by the presence of the autochthonous microbiota. The present findings that Aer. hydrophila survives very poorly in several drinking waters is of utmost importance towards public health and arises questions about the mechanisms involved.

Gene transfer in magnetic bacteria: transposon mutagenesis and cloning of genomic DNA fragments required for magnetosome synthesis.

Broad-host-range IncP and IncQ plasmids have been transferred to the aerobic magnetic bacterium Aquaspirillum sp. strain AMB-1. Conjugal matings with Escherichia coli S17-1 allowed high-frequency transfer of the RK2 derivative pRK415 (4.5 x 10(-3) transconjugant per recipient cell) and the RSF1010 derivative pKT230 (3.0 x 10(-3) transconjugant per recipient). These plasmids successfully formed autonomous replicons in transconjugants and could be isolated and transformed back into E. coli, illustrating their potential as shuttle vectors. A mobilizable plasmid containing transposon Tn5 was transferred to Aquaspirillum sp. strain AMB-1 and also to the obligately microaerophilic magnetic bacterium Aquaspirillum magnetotacticum MS-1. Five nonmagnetic kanamycin-resistant mutants of Aquaspirillum sp. strain AMB-1 in which Tn5 was shown to be integrated into the chromosome were obtained. Different genomic fragments containing the mutagenized regions were cloned into E. coli. Two genomic fragments were restriction mapped, and the site of Tn5 insertion was determined. They were shown to be identical, although derived from independent transposon insertions. One of these clones was found to hybridize strongly to regions of the A. magnetotacticum MS-1 chromosome. This is the first report of gene transfer in a magnetic bacterium.

Identification of a promoter dependent on NifA and sigma 54 upstream of nifH in Azospirillum lipoferum.

Southern hybridization experiments strongly indicate that the regulatory region of the Azospirillum lipoferum nifH gene is located on a cloned 1.1 kb BamHI-XhoI restriction fragment. By cloning this fragment into a promoter-probe plasmid in Escherichia coli, a promoter was identified oriented towards the nifH gene. Using a set of several bacterial strains and plasmids, both NifA and the alternative sigma factor, sigma 54, from Klebsiella pneumoniae were shown to be required for the induction of the assumed nifH promoter in this particular heterologous system. However, NtrC from K. pneumoniae did not stimulate this promoter. No other promoter activity was detected in the direction opposite to the identified promoter, indicating that the transcription of the adjacent nifJ gene cannot be initiated from the 1.1 kb BamHI-XhoI fragment. Thus, the genes nifH and nifJ in A. lipoferum cannot be oriented divergently, in contrast to the situation in several other nitrogen-fixing bacteria.

Expression of the Agrobacterium tumefaciens chvB virulence region in Azospirillum spp.

Inner membranes of Azospirillum brasilense incubated with UDP-glucose were unable to synthesize beta-(1-2) glucan and lacked the 235-kilodalton intermediate protein known to be involved in the synthesis of beta-(1-2) glucan in Agrobacterium tumefaciens and Rhizobium meliloti. Inner membranes of A. brasilense strains carrying a cosmid containing the chromosomal virulence genes chvA and chvB of Agrobacterium tumefaciens formed beta-(1-2) glucan in vitro and synthesized the 235-kilodalton intermediate protein. No DNA homology to the chvB region was found in different wild-type strains of A. brasilense, but the introduction of a cosmid containing the Agrobacterium tumefaciens chvA and chvB regions yielded strains in which DNA hybridization with the chvB region was detected, provided that the strains were grown under an antibiotic selective pressure.

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene.

A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxy-terminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.

Isolation and characterization of Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutations.

The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.

Construction of a gene library from Azospirillum brasilense and characterization of a recombinant containing the nif structural genes.

1. We have constructed a gene library, from Azospirillum brasilense using the vector EMBL4. 2. A recombinant containing the nif structural genes from A. brasilense was isolated and characterized. This recombinant contains a DNA insert of about 15 kilobases (kb) which gives rise to five fragments after cleavage with EcoRI. Only one of the DNA fragments (6.5 kb) hybridized to the nifHDK genes of Klebsiella pneumoniae. 3. The organization of the nif genes in this DNA fragment was determined using different DNA segments containing the nifH, nifK or nifD genes of K. pneumoniae as probes.

Construction of a gene library from Azospirillum brasilense and characterization of a recombinant containing the nif structural genes

1.We have constructed a gene library, from Azospirillum brasilense using the vector EMBL4. 2. A recombinant containing the nif structural genes from A. brasilense was isolated and characterized. This recombinant contains a DNA insert of about 15 kilobases (KB) which gives rise to five fragments after cleavage with EcoRI. Only one of the DNA fragments (6.5 Kb) hybridized to the nifHDK genes of Klebsiella pneumoniae. 3 The organization of the nif genes in this DNA fragment was determined using different DNA segments containing the nifH, nifK or nifD genes of K. pneumoniae as probes

Contenido de plásmidos em cepas de Azospirillum sp. / Plasmid content in Azospirillum strains

En este trabajo se describe el contenido de plásmidos de 27 cepas de A. brasilense y A. lipoferum aisladas de diferentes zonas del país y de distintas especies vegetales y se describen características de los métodos de extracción de plásmidos de alto peso molecular empleados. Casi todas las cepas presentaron entre uno y cuatro plásmidos, todos de alto peso molecular. Uno de ellos, de aproximadamente 100 Megadaltons, se encuentra en el 44% de las cepas analizadas. Aún no hay evidencias en cuanto a la información genética codificada por ellos

Contenido de plásmidos em cepas de Azospirillum sp. / Plasmid content in Azospirillum strains

En este trabajo se describe el contenido de plásmidos de 27 cepas de A. brasilense y A. lipoferum aisladas de diferentes zonas del país y de distintas especies vegetales y se describen características de los métodos de extracción de plásmidos de alto peso molecular empleados. Casi todas las cepas presentaron entre uno y cuatro plásmidos, todos de alto peso molecular. Uno de ellos, de aproximadamente 100 Megadaltons, se encuentra en el 44% de las cepas analizadas. Aún no hay evidencias en cuanto a la información genética codificada por ellos (AU)

Cloning and characterization of the glnA gene of Azospirillum brasilense Sp7.

A plasmid which, by complementation, restored a Gln+Nif+ phenotype to the Gln-Nif- Azospirillum brasilense mutant 7029, was isolated from a gene bank of total DNA of A. brasilense Sp7 (ATCC 29145) constructed in the broad host range vector pVK100. This plasmid contained the structural gene (glnA) for glutamine synthetase. The glnA gene was mapped by Tn5 insertion and DNA hybridization with a Klebsiella pneumoniae glnA probe. The direction of transcription of glnA was determined. The glnA product was identified as a 50-Kd polypeptide which could be adenylylated in Escherichia coli, and glutamine synthetase activity was characterized in E. coli. Plasmids containing the glnA gene restored glutamine-independent growth and a Nif+ phenotype to Gln-Nif- and Gln-Nifc mutants of Azospirillum.

Restriction endonucleases in Azospirillum.

Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions.