Genes required for phosphosphingolipid formation in Caulobacter crescentus contribute to bacterial virulence.

Sphingolipids are ubiquitous in membranes of eukaryotes and are associated with important cellular functions. Although sphingolipids occur scarcely in bacteria, for some of them they are essential and, in other bacteria, they contribute to fitness and stability of the outer membrane, such as in the well-studied α-proteobacterium Caulobacter crescentus. We previously defined five structural genes for ceramide synthesis in C. crescentus, among them the gene for serine palmitoyltransferase, the enzyme that catalyzes the committed step of sphingolipid biosynthesis. Other mutants affected in genes of this same genomic region show cofitness with a mutant deficient in serine palmitoyltransferase. Here we show that at least two phosphosphingolipids are produced in C. crescentus and that at least another six gene products are needed for the decoration of ceramide upon phosphosphingolipid formation. All eleven genes participating in phosphosphingolipid formation are also required in C. crescentus for membrane stability and for displaying sensitivity towards the antibiotic polymyxin B. The genes for the formation of complex phosphosphingolipids are also required for C. crescentus virulence on Galleria mellonella insect larvae.

Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

ExoS/ChvI Two-Component Signal-Transduction System Activated in the Absence of Bacterial Phosphatidylcholine.

Sinorhizobium meliloti contains the negatively charged phosphatidylglycerol and cardiolipin as well as the zwitterionic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) as major membrane phospholipids. In previous studies we had isolated S. meliloti mutants that lack PE or PC. Although mutants deficient in PE are able to form nitrogen-fixing nodules on alfalfa host plants, mutants lacking PC cannot sustain development of any nodules on host roots. Transcript profiles of mutants unable to form PE or PC are distinct; they differ from each other and they are different from the wild type profile. For example, a PC-deficient mutant of S. meliloti shows an increase of transcripts that encode enzymes required for succinoglycan biosynthesis and a decrease of transcripts required for flagellum formation. Indeed, a PC-deficient mutant is unable to swim and overproduces succinoglycan. Some suppressor mutants, that regain swimming and form normal levels of succinoglycan, are altered in the ExoS sensor. Our findings suggest that the lack of PC in the sinorhizobial membrane activates the ExoS/ChvI two-component regulatory system. ExoS/ChvI constitute a molecular switch in S. meliloti for changing from a free-living to a symbiotic life style. The periplasmic repressor protein ExoR controls ExoS/ChvI function and it is thought that proteolytic ExoR degradation would relieve repression of ExoS/ChvI thereby switching on this system. However, as ExoR levels are similar in wild type, PC-deficient mutant and suppressor mutants, we propose that lack of PC in the bacterial membrane provokes directly a conformational change of the ExoS sensor and thereby activation of the ExoS/ChvI two-component system.

Five structural genes required for ceramide synthesis in Caulobacter and for bacterial survival.

Sphingolipids are essential and common membrane components in eukaryotic organisms, participating in many important cellular functions. Only a few bacteria are thought to harbour sphingolipids in their membranes, among them the well-studied α-proteobacterium Caulobacter crescentus, a model organism for asymmetric cell division and cellular differentiation. Here, we report that C. crescentus wild type produces several molecular species of dihydroceramides, which are not produced in a mutant lacking the structural gene for serine palmitoyltransferase (spt). Whereas growth of a spt-deficient mutant and wild type are indistinguishable during the exponential phase of growth, survival of the spt-deficient mutant is much reduced, in comparison with wild type, during stationary phase of growth, especially at elevated temperatures. The structural gene for spt is located within a genomic cluster, comprising another 16 genes and which, like spt, are important for fitness of C. crescentus. Mutants deficient in genes linked to spt by high cofitness were unable to produce dihydroceramide or to survive in stationary phase of growth at elevated temperatures. At least five structural genes are required for dihydroceramide biosynthesis in C. crescentus and sphingolipid biosynthesis is needed for survival of this bacterium and the integrity of its outer membrane.

Paraburkholderia lycopersici sp. nov., a nitrogen-fixing species isolated from rhizoplane of Lycopersicon esculentum Mill. var. Saladette in Mexico.

A survey of our in-house bacterial collection identified a group of six strains isolated from the tomato rhizoplane that possessed 16S rRNA gene sequences with 98.2% sequence similarity to Paraburkholderia pallida, suggesting that these strains represented a novel species. Multilocus sequence analysis using gltB, lepA and recA gene sequences showed the clustering of the strains and the BOX-PCR patterns were similar among these strains. The average nucleotide identity and the DNA-DNA virtual hybridization of strain TNe-862T was <89% and <34%, respectively, to the genomes of any sequenced Paraburkholderia species. The genome of strain TNe-862T possessed all the genes necessary for nitrogen fixation and biosynthesis of indoleacetic acid and antimicrobials terpenes, phosphonates and bacteriocins. It also contained genes for metal resistance, xenobiotic degradation, and hydrolytic enzymes such as a putative chitinase and isoamylase. Even though the strain contained potential genes for degradation of cellulose and starch, the bacterium was unable to utilize these substrates in culture medium. The genome encoded flagella and pili as well as multiple chemotaxis systems. In addition, genes encoding for the type I, II, IV, V and VI secretion systems were also present. The strains grow up to 42°C and 5% NaCl. The optimum growth pH was 8. The major cellular fatty acids were C16:0 and C18:1 ω7c. Based on this polyphasic analysis, these strains represent a novel species in the genus Paraburkholderia, for which the name Paraburkholderia lycopersici sp. nov. is proposed. The type strain is TNe-862T (=LMG 26415T=CIP 110323T).

Ornithine Lipids in Burkholderia spp. Pathogenicity.

The genus Burkholderia sensu lato is composed of a diverse and metabolically versatile group of bacterial species. One characteristic thought to be unique for the genus Burkholderia is the presence of two forms each (with and without 2-hydroxylation) of the membrane lipids phosphatidylethanolamine (PE) and ornithine lipids (OLs). Here, we show that only Burkholderia sensu stricto strains constitutively form OLs, whereas all other analyzed strains belonging to the Burkholderia sensu lato group constitutively form the two forms of PE, but no OLs. We selected two model bacteria to study the function of OL in Burkholderia sensu lato: (1) Burkholderia cenocepacia wild-type which constitutively forms OLs and its mutant deficient in the formation of OLs and (2) Robbsia andropogonis (formerly Burkholderia andropogonis) which does not form OL constitutively, and a derived strain constitutively forming OLs. Both were characterized under free-living conditions and during pathogenic interactions with their respective hosts. The absence of OLs in B. cenocepacia slightly affected bacterial growth under specific abiotic stress conditions such as high temperature and low pH. B. cenocepacia lacking OLs caused lower mortality in Galleria mellonella larvae while R. andropogonis constitutively forming OLs triggers an increased formation of reactive oxygen species immediately after infection of maize leaves, suggesting that OLs can have an important role during the activation of the innate immune response of eukaryotes.

Burkholderia caballeronis sp. nov., a nitrogen fixing species isolated from tomato (Lycopersicon esculentum) with the ability to effectively nodulate Phaseolus vulgaris.

During a survey of Burkholderia species with potential use in agrobiotechnology, a group of 12 strains was isolated from the rhizosphere and rhizoplane of tomato plants growing in Mexico (Nepantla, Mexico State). A phylogenetic analysis of 16S rRNA gene sequences showed that the strains are related to Burkholderia kururiensis and Burkholderia mimosarum (97.4 and 97.1 %, respectively). However, they induced effective nitrogen-fixing nodules on roots of Phaseolus vulgaris. Based on polyphasic taxonomy, the group of strains represents a novel species for which the name Burkholderia caballeronis sp. nov. is proposed. The type species is TNe-841(T) (= LMG 26416(T) = CIP 110324(T)).

Phylogenetic analysis of burkholderia species by multilocus sequence analysis.

Burkholderia comprises more than 60 species of environmental, clinical, and agro-biotechnological relevance. Previous phylogenetic analyses of 16S rRNA, recA, gyrB, rpoB, and acdS gene sequences as well as genome sequence comparisons of different Burkholderia species have revealed two major species clusters. In this study, we undertook a multilocus sequence analysis of 77 type and reference strains of Burkholderia using atpD, gltB, lepA, and recA genes in combination with the 16S rRNA gene sequence and employed maximum likelihood and neighbor-joining criteria to test this further. The phylogenetic analysis revealed, with high supporting values, distinct lineages within the genus Burkholderia. The two large groups were named A and B, whereas the B. rhizoxinica/B. endofungorum, and B. andropogonis groups consisted of two and one species, respectively. The group A encompasses several plant-associated and saprophytic bacterial species. The group B comprises the B. cepacia complex (opportunistic human pathogens), the B. pseudomallei subgroup, which includes both human and animal pathogens, and an assemblage of plant pathogenic species. The distinct lineages present in Burkholderia suggest that each group might represent a different genus. However, it will be necessary to analyze the full set of Burkholderia species and explore whether enough phenotypic features exist among the different clusters to propose that these groups should be considered separate genera.

Transfer of Wautersia numazuensis to the genus Cupriavidus as Cupriavidus numazuensis comb. nov.

Phylogenetic analysis of the 16S rRNA gene sequences of strains TE26(T) and K6 belonging to Wautersia numazuensis Kageyama et al. 2005 showed the strains to be deeply intermingled among the species of the genus Cupriavidus. The comparison showed that strain TE26(T) was closely related to the type strains of Cupriavidus pinatubonensis (99.1 % 16S rRNA gene sequence similarity), C. basilensis (98.7 %), C. necator (98.7 %) and C. gilardii (98.0 %). However, DNA-DNA hybridization experiments (less than 20 % relatedness) demonstrated that strain TE26(T) is different from these Cupriavidus species. A comparative phenotypic and chemotaxonomic analysis (based on fatty acid profiles) in combination with the 16S rRNA gene sequence phylogenetic analysis and the DNA-DNA hybridization results supported the incorporation of Wautersia numazuensis into the genus Cupriavidus as Cupriavidus numazuensis comb. nov.; the type strain is TE26(T) (=LMG 26411(T) =DSM 15562(T) = CIP 108892(T)).

Cupriavidus alkaliphilus sp. nov., a new species associated with agricultural plants that grow in alkaline soils.

A group of 20 bacterial strains was isolated from the rhizosphere of different agricultural plants growing in alkaline soils in the northeast of Mexico. The phylogenetic analysis of the 16S rRNA gene sequence from four strains showed that this novel group belonged to the Cupriavidus genus, with C. taiwanensis (∼98.9%) and C. necator (∼98.8%) as the closest species. However, DNA-DNA reassociation values were less than 20%. The novel group did not fix nitrogen and lacked nifH and nodA genes, unlike C. taiwanensis. Whole-cell protein patterns were highly similar among the 20 strains but different from the closest Cupriavidus species. BOX-PCR patterns were distinct among the 20 strains but also differed from other Cupriavidus type species. The major cellular fatty acids from strains ASC-732(T) and SLV-2362 were C(16:0), C(18:1) ω7c/12t/9t and C(16:1) ω7c and/or C(15:0) iso 2OH. The major polar lipids consisted of phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, 2-hydroxylated-phosphatidylethanolamine and an unknown aminolipid. The DNA G+C content of strain ASC-732(T) was 66.8mol%. All 20 strains grew in the presence of 5-10mgmL(-1) arsenic, 1mgmL(-1) zinc, and 0.1mgmL(-1) copper. Consequently, the group of strains was considered to represent a novel species for which the name Cupriavidus alkaliphilus sp. nov. is proposed. The type strain is ASC-732(T) (=LMG 26294(T)=CIP 110330(T)).

Cupriavidus and Burkholderia species associated with agricultural plants that grow in alkaline soils.

The presence of Burkholderia, Cupriavidus, and Ralstonia species in northeastern Mexico was investigated. An analysis of the root surrounding soil from different agricultural plants led to the isolation of Burkholderia and Cupriavidus species but no Ralstonia strains. Most Cupriavidus species were unknown and grouped into two clusters according to ARDRA profiles. The 16S rRNA sequence analysis showed that the Cupriavidus isolates were highly related among them and with different Cupriavidus species with validated names. However, SDS-PAGE profiles were distinct among the different ARDRA profiles and to other Cupriavidus species examined, suggesting new species in the genus. This shows that Cupriavidus is more widely associated with plants than previously appreciated. The BCC isolate was 99% similar to B. cenocepacia by recA sequence analysis. Additionally, most Cupriavidus strains from the two largest groups grew on media containing up to 0.1 mg/ml of copper, 10.0 mg/ml arsenic and 1.0 mg/ml zinc. Burkholderia strains grew on media containing up to 10.0 mg/ml zinc, 5.0 mg/ml arsenic and 0.1 mg/ml copper.

Pseudomonas cuatrocienegasensis sp. nov., isolated from an evaporating lagoon in the Cuatro Cienegas valley in Coahuila, Mexico.

Nine Gram-negative, rod-shaped, non-spore-forming isolates with identical or very similar repetitive-sequence-based PCR profiles were recovered from an evaporative lagoon in Mexico. Two strains, designated 1N(T) and 3N, had virtually identical 16S rRNA gene sequences and, on the basis of these sequences, were identified as members of the genus Pseudomonas, with Pseudomonas peli R-20805(T) as the closest relative. All nine isolates had practically identical whole-cell protein profiles. The major fatty acids [C(16 : 0,) C(18 : 1)omega7c and summed feature a (C(16 : 1)omega7 and/or C(16 : 1)omega6c)] of strains 1N(T) and 3N supported their affiliation with the genus Pseudomonas. The DNA-DNA reassociation values with respect to P. peli LMG 23201(T) and other closely related Pseudomonas species were <15 %. Physiological and biochemical tests allowed phenotypic differentiation of the strains analysed, including strain 1N(T), from the five phylogenetically closest Pseudomonas species. On the basis of the data obtained by using this polyphasic taxonomic approach, the nine strains represent a novel species, for which the name Pseudomonas cuatrocienegasensis sp. nov. is proposed. The type strain is 1N(T) (=LMG 24676(T)=CIP 109853(T)).