Glucose Metabolism in Batch and Continuous Cultures of Gluconacetobacter diazotrophicus PAL 3

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]–linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N₂ or NH₃). Its synthesis was stimulated by conditions of high energetic demand (i.e., N₂-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N₂-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)–linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.     

Respiratory system of Gluconacetobacter diazotrophicus PAL5. Evidence for a cyanide-sensitive cytochrome bb and cyanide-resistant cytochrome ba quinol oxidases

In highly aerobic environments, Gluconacetobacter diazotrophicus uses a respiratory protection mechanism to preserve nitrogenase activity from deleterious oxygen. Here, the respiratory system was examined in order to ascertain the nature of the respiratory components, mainly of the cyanide sensitive and resistant pathways. The membranes of G. diazotrophicus contain Q(10), Q(9) and PQQ in a 13:1:6.6 molar ratios. UV(360 nm) photoinactivation indicated that ubiquinone is the electron acceptor for the dehydrogenases of the outer and inner faces of the membrane. Strong inhibition by rotenone and capsaicin and resistance to flavone indicated that NADH-quinone oxidoreductase is a NDH-1 type enzyme. KCN-titration revealed the presence of at least two terminal oxidases that were highly sensitive and resistant to the inhibitor. Tetrachorohydroquinol was preferentially oxidized by the KCN-sensitive oxidase. Neither the quinoprotein alcohol dehydrogenase nor its associated cytochromes c were instrumental components of the cyanide resistant pathway. CO-difference spectrum and photodissociation of heme-CO compounds suggested the presence of cytochromes b-CO and a(1)-CO adducts. Air-oxidation of cytochrome b (432 nm) was arrested by concentrations of KCN lower than 25 microM while cytochrome a(1) (442 nm) was not affected. A KCN-sensitive (I(50)=5 microM) cytochrome bb and a KCN-resistant (I(50)=450 microM) cytochrome ba quinol oxidases were separated by ion exchange chromatography.     

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.     

The respiratory system and diazotrophic activity of Acetobacter diazotrophicus PAL5

The characteristics of the respiratory system of Acetobacter diazotrophicus PAL5 were investigated. Increasing aeration (from 0.5 to 4.0 liters of air min(-1) liter of medium(-1)) had a strong positive effect on growth and on the diazotrophic activity of cultures. Cells obtained from well-aerated and diazotrophically active cultures possessed a highly active, membrane-bound electron transport system with dehydrogenases for NADH, glucose, and acetaldehyde as the main electron donors. Ethanol, succinate, and gluconate were also oxidized but to only a minor extent. Terminal cytochrome c oxidase-type activity was poor as measured by reduced N, N,N,N'-tetramethyl-p-phenylenediamine, but quinol oxidase-type activity, as measured by 2,3,5,6-tetrachloro-1,4-benzenediol, was high. Spectral and high-pressure liquid chromatography analysis of membranes revealed the presence of cytochrome ba as a putative oxidase in cells obtained from diazotrophically active cultures. Cells were also rich in c-type cytochromes; four bands of high molecular mass (i.e., 67, 56, 52, and 45 kDa) were revealed by a peroxidase activity stain in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KCN inhibition curves of respiratory oxidase activities were biphasic, with a highly resistant component. Treatment of membranes with 0.2% Triton X-100 solubilized c-type cytochromes and resulted in a preparation that was significantly more sensitive to cyanide. Repression of diazotrophic activity in well-aerated cultures by 40 mM (NH(4))(2)SO(4) caused a significant decrease of the respiratory activities. It is noteworthy that the levels of glucose dehydrogenase and putative oxidase ba decreased 6. 8- and 10-fold, respectively. In these cells, a bd-type cytochrome seems to be the major terminal oxidase. Thus, it would seem that glucose dehydrogenase and cytochrome ba are key components of the respiratory system of A. diazotrophicus during aerobic diazotrophy.     

Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km^R -mutant strains in a medium without iron supplementation and in a medium containing 2, 2′-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km^R -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.     

Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5™<KAN-2>Tnp Transposome™ (Epicentre). Up to 1 × 10⁶ mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

A comparative proteomic analysis of Gluconacetobacter diazotrophicus PAL5 at exponential and stationary phases of cultures in the presence of high and low levels of inorganic nitrogen compound

A proteomic view of G. diazotrophicus PAL5 at the exponential (E) and stationary phases (S) of cultures in the presence of low (L) and high levels (H) of combined nitrogen is presented. The proteomes analyzed on 2D-gels showed 131 proteins (42E+32S+29H+28L) differentially expressed by G. diazotrophicus, from which 46 were identified by combining mass spectrometry and bioinformatics tools. Proteins related to cofactor, energy and DNA metabolisms and cytoplasmic pH homeostasis were differentially expressed in E growth phase, under L and H conditions, in line with the high metabolic rate of the cells and the low pH of the media. Proteins most abundant in S-phase cells were stress associated and transporters plus transferases in agreement with the general phenomenon that binding protein-dependent systems are induced under nutrient limitation as part of hunger response. Cells grown in L condition produced nitrogen-fixation accessory proteins with roles in biosynthesis and stabilization of the nitrogenase complex plus proteins for protection of the nitrogenases from O(2)-induced inactivation. Proteins of the cell wall biogenesis apparatus were also expressed under nitrogen limitation and might function in the reshaping of the nitrogen-fixing G. diazotrophicus cells previously described. Genes whose protein products were detected in our analysis were mapped onto the chromosome and, based on the tendency of functionally related bacterial genes to cluster, we identified genes of particular pathways that could be organized in operons and are co-regulated. These results showed the great potential of proteomics to describe events in G. diazotrophicus cells by looking at proteins expressed under distinct growth conditions.     

Modeling the quorum sensing regulatory network of human-pathogenic Pseudomonas aeruginosa

The biochemical network underlying quorum sensing in human-pathogenic Pseudomonas aeruginosa is one of the best characterized. Mathematical modeling is required to untangle the complexity of its architecture and dynamics. We present a qualitative model of the P. aeruginosa quorum-sensing network including interactions between the las and rhl modules, the signaling molecule PQS and the regulatory proteins Mvfr and VfR. Simulations exemplify the model to reproduce natural network behavior and suggest quorum-sensing responses to pharmacological interference.     

从进化谈细菌细胞间的群体感应信号传递

传统观念认为细菌是一种个体的、非社会性的生物体。近年来的研究表明细菌可以产生化学信号并通过它们实现细菌间信息传递。细菌的群体感应调节系统(Quorum sensing,QS)调节着个体细胞之间的相互合作,使其表现出类似多细胞的群体行为。文章以近年来的一些最新研究进展为基础,在了解细菌间的信息传递系统的基础上,从进化角度讨论了QS系统的遗传产生过程,探讨了细菌细胞间的相互作用。细菌间的信息交流是一种动态的过程,受到了环境中的营养物质的水平、温度、pH等多种因素的影响。作者推测细菌信号传递系统的进化是受到环境条件以及基因交换、所在微生物群体变化等因素影响下的一种不断变化的动态过程,这也许有别于动植物这类的高等生物的进化过程。这种动态的变化过程也就暗示:从长远来看,信息传递系统中的偷机者只是在一定条件下的暂时存在。     

Essential role of the czc determinant for cadmium, cobalt and zinc resistance in Gluconacetobacter diazotrophicus PAl 5

The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl2·H2O, CoCl2·6H2O and ZnCl2. Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5 in the presence of ZnO revealed that the mutant GDP30H3 was unable to grow in the presence of the compound. This mutant was also highly sensitive to CdCl2·H2O, CoCl2·6H2O and ZnCl2. Molecular characterization established that the mutation affected the czcA gene, which encodes a protein involved in metal efflux. In silico analysis showed that czcA is a component of the czcCBARS operon together with four other genes. This work provides evidence of the high tolerance of G. diazotrophicus PAl 5 to heavy metals and that czc is a determinant for metal resistance in this bacterium.     

多重耐药铜绿假单胞菌群体感应系统与主动外排基因表达的研究

目的研究临床多重耐药铜绿假单胞菌群体感应(QS)系统与主动外排泵MexAB-OprM系统基因表达水平与抗生素耐药关系。方法收集苏州市立医院和上海市江湾医院2011年2月至6月间临床标本中分离的铜绿假单胞菌,定量分析细菌生物被膜形成能力;MIC法检测细菌抗生素耐药性,用多重聚合酶链反应(PCR)扩增群体感应系统lasI、lasR及主动外排泵系统mexA基因,实时定量逆转录RT-PCR检测lasI、lasR和mexA基因的相对表达量。结果临床样本分离出84株铜绿假单胞菌,其中产生物被膜菌58株,占比69%;多重耐药菌共24株,占比28.6%;多重耐药菌株中产生物被膜有11株,占45.8%;多重耐药菌中mexA基因表达上调有18株,占75%;lasI基因表达上调有8株,占33.3%。结论多重耐药菌株的生物被膜形成率显著低于非多重耐药组,多重耐药铜绿假单胞菌的主动外排泵MexAB-OprM系统基因表达出现显著上调,生物被膜菌的lasI基因表达显著上调而lasR基因的表达无明显变化。     

Co-regulation of Xanthomonas campestris virulence by quorum sensing and a novel two-component regulatory system RavS/RavR

Xanthomonas campestris pv. campestris ( Xcc ) is known to regulate virulence through a quorum-sensing mechanism. Detection of the quorum-sensing signal DSF by sensor RpfC leads to activation of the response regulator RpfG, which influences virulence by degrading cyclic-di-GMP and by subsequent increasing expression of the global regulator Clp. In this study, we show that mutation of a response regulator RavR containing the GGDEF–EAL domains decreases Xcc virulence factor production . The functionality of RavR is dependent on its EAL domain-associated cyclic-di-GMP phosphodiesterase activity. Deletion of a multidomain sensor gene ravS , which shares the same operon with ravR , results in similar phenotype changes as the ravR mutant. In addition, the sensor mutant phenotypes can be rescued by in trans expression of the response regulator, supporting the notion that RavS and RavR constitute a two-component regulatory system. Significantly, mutation of either the PAS domain or key residues of RavS implicated in sensing low-oxygen tension abrogates the sensor activity in virulence regulation. Moreover, similar to the DSF signalling system, RavS/RavR regulates virulence gene expression through the global regulator Clp. These results outline a co-regulation mechanism that allows Xcc to integrate population density and environmental cues to modulate virulence factor production and adaptation.     

Clinical strains of Burkholderia cepacia: characteristic and detection of the components in the quorum sensing regulatory system

Described in the paper are characteristics of B. cepacia clinical strains isolated from patients at Moscow hospitals. The strains were investigated for the presence of proteolytic, chitinolytic, hemolytic and lipase activities as well as for presence of components of the "Quorum sensing" gene activity regulatory system by using biological test-systems and in the polymerase chain reaction with primers to genes cepI and cepR.     

Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing

Quorum sensing is a mechanism of cell‐to‐cell communication that allows bacteria to coordinately regulate gene expression in response to changes in cell‐population density. At the core of the Vibrio cholerae quorum‐sensing signal transduction pathway reside four homologous small RNAs (sRNAs), named the quorum regulatory RNAs 1–4 (Qrr1–4). The four Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild‐type quorum‐sensing behaviour. Here, we show that the combined action of two feedback loops, one involving the sRNA‐activator LuxO and one involving the sRNA‐target HapR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr1–4 sRNA pool and provides the molecular mechanism underlying sRNA redundancy. The dosage compensation mechanism is exquisitely sensitive to small perturbations in Qrr levels. Precisely maintained Qrr levels are required to direct the proper timing and correct patterns of expression of quorum‐sensing‐regulated target genes.