Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions.

We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R. meliloti ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation. DNA sequence analysis showed that R. meliloti NtrA is 38% homologous with Klebsiella pneumoniae NtrA. Subcloning and complementation analysis suggested that the R. meliloti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.     

Presence of flagella in Pseudomonas putida is dependent on the ntrA (rpoN) gene

Summary An ntrA (rpoN) mutant of Pseudomonas putida, in which the gene was insertionally inactivated, was constructed. The mutant cells did not have flagella, thus accounting for their poor motility. The mutant phenotype was complemented by introduction of the intact ntrA gene.     

Virulence of the phytopathogen Pseudomonas syringae pv. maculicola is rpoN dependent

We cloned the rpoN (ntrA and glnF) gene encoding sigma(54) from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the sigma(54) protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km(r). In contrast to wild-type ES4326, ES4326 rpoN::Km(r) was nonmotile and could not utilize nitrate, urea, C(4)-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN::Km(r) did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Km(r) carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN::Km(r) partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN::Km(r) did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.     

The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins

Summary The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing>50% homology and an intervening sequence of <10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.     

Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili

We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.     

Pseudomonas putida Which Can Grow in the Presence of Toluene

A Pseudomonas putida strain able to grow in the presence of more than 50% toluene was isolated from soil. The strain was tolerant of other toxic solvents, including aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, alcohols, and ethers. The stability of the solvent tolerance of strain IH-2000 was stimulated by addition of Mg²⁺ and Ca²⁺ to the medium containing toluene.     

Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida

Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioaccumulation of Germanium by Pseudomonas putida in the Presence of Two Selected Substrates

The uptake of germanium by Pseudomonas putida ATCC 33015 was studied in the presence of catechol or acetate or both as representative substrates differing in their ability to form complexes with this element. The bacteria were taken from a batch culture grown on acetate as the sole carbon source. Cells introduced into a medium containing germanium and either catechol or a mixture of catechol and acetate accumulated germanium in a biphasic way. After a lower level of accumulation that corresponded to the value obtained in the presence of acetate was reached, a further increase in the germanium content up to a higher saturation level was observed. The appearance of the second step of accumulation, which corresponded to the linear degradation of catechol, proved that catechol facilitated the transport of germanium into the cells through the nonspecific uptake of the germanium-catechol complex by an inducible catechol transport system.     

Selected Pseudomonas putida Strains Able To Grow in the Presence of High Butanol Concentrations

Adapted Pseudomonas putida strains grew in the presence of up to 6% (vol/vol) butanol, the highest reported butanol concentration tolerated by a microbe. P. putida might be an alternative host for biobutanol production, overcoming the primary limitation of currently used strains—insufficient product titers due to low butanol tolerance.The focus of biofuel production research has recently shifted from ethanol to bioenergy carriers that are more compatible with existing infrastructure (e.g., refineries, transport, and car engines). At the forefront is n-butanol (hereafter referred to as butanol) for which large-scale production processes have been implemented (16, 35). Existing fermentations, however, are limited in energetically attractive butanol titers, because butanol inhibits microbial growth at concentrations above 16 g/liter (2, 10). As reported for other organic solvents with low logarithm of the partition coefficient in a two-phase octanol/water system (log P_ow), this toxicity is due primarily to accumulation of butanol (log P_ow, 0.8) in the cell membrane and subsequent impairment (4, 17, 30, 33). With the maximum aqueous solubility of 0.97 M (8.8% [vol/vol]), the maximum membrane concentration of butanol was calculated to be 1.59 M (17), spotlighting its potential toxicity. The low achievable butanol titers have necessitated large reactor volumes, resulting in high purification costs (8, 15). Recent metabolic engineering strategies for improving biobutanol fermentation have focused on maximization of butanol production rates (10, 19), reducing the levels of by-products (20), finding alternative substrates (20), or finding alternative hosts (2, 12, 21, 31). However, recently engineered microbial strains (1, 14) have not overcome butanol toxicity.High organic solvent concentrations are tolerated by strains of the bacterial species Pseudomonas putida reported to grow in a second phase of octanol (25), toluene (13), or styrene (32). This suggests that solvent-tolerant P. putida strains withstand high butanol titers and therefore warrant exploitation as host for butanol production. Indeed, viable solvent-tolerant P. putida S12 cells were observed at butanol concentrations of up to 10% (vol/vol) by live-dead staining and fluorescence microscopy (5) (see supplemental material). We used growth as the parameter of interest, because growth in the presence of butanol directly indicates the potential of selected P. putida strains as hosts for recombinant butanol production.Three solvent-tolerant P. putida strains, DOT-T1E (23), S12 (32), and Pseudomonas sp. strain VLB120 (18), and the solvent-sensitive P. putida reference strain KT2440 (24) were examined for their ability to grow in the presence of butanol. Toxicity assays were performed in 96-well microtiter plates (System Duetz [7]) at 30°C and 300 rpm using glucose-supplemented LB and M9 media (with 10 and 5 g/liter glucose, respectively) (26). Higher glucose concentrations in LB medium did not increase butanol tolerance (data not shown). Butanol was added in all experiments to cells in the mid-exponential phase. Cell growth was monitored by changes in optical density, and substrate and butanol concentrations were analyzed by high-pressure liquid chromatography (Trentec 308R-Gel.H; VWR Hitachi). Comparable low butanol concentrations were withstood by all P. putida strains, with butanol tolerance highly dependent on the medium composition (Table ​(Table1).1). Growth was observed at butanol concentrations up to 3% (vol/vol), occurring in a culture of Pseudomonas sp. strain VLB120 using glucose-supplemented LB medium.

TABLE 1.

Tolerated butanol concentrations in different growth media

A new type of flagellin gene in Pseudomonas putida

Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.     

PanB is involved in nicotine metabolism in Pseudomonas putida

A nicotine-sensitive mutant was generated from the nicotine-degrading bacterium, Pseudomonas putida strain J5, by mini-Tn5 transposon mutagenesis. This mutant was unable to grow with nicotine as the sole carbon source but could grow with glucose. Sequence analysis showed that the Tn5 transposon inserted at the site of the ketopantoate hydroxymethyltransferase gene (panB), which had 54% identity to PanB in Escherichia coli K-12. In-frame deletion of the panB gene abolished the nicotine-degrading ability of strain J5, while complementation with panB from P. putida J5 and E. coli K-12 restored the degrading activity of the mutant to the wild-type level. These results suggest that ketopantoate hydroxymethyltransferase is a crucial enzyme in nicotine metabolism in P. putida J5.