Global features of the Alcanivorax borkumensis SK2 genome

The global feature of the completely sequenced Alcanivorax borkumensis SK2 type strain chromosome is its symmetry and homogeneity. The origin and terminus of replication are located opposite to each other in the chromosome and are discerned with high signal to noise ratios by maximal oligonucleotide usage biases on the leading and lagging strand. Genomic DNA structure is rather uniform throughout the chromosome with respect to intrinsic curvature, position preference or base stacking energy. The orthologs and paralogs of A. borkumensis genes with the highest sequence homology were found in most cases among γ-Proteobacteria, with Acinetobacter and P. aeruginosa as closest relatives. A. borkumensis shares a similar oligonucleotide usage and promoter structure with the Pseudomonadales. A comparatively low number of only 18 genome islands with atypical oligonucleotide usage was detected in the A. borkumensis chromosome. The gene clusters that confer the assimilation of aliphatic hydrocarbons, are localized in two genome islands which were probably acquired from an ancestor of the Yersinia lineage, whereas the alk genes of Pseudomonas putida still exhibit the typical Alcanivorax oligonucleotide signature indicating a complex evolution of this major hydrocarbonoclastic trait.     

Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2

Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. To identify the genes responsible for alkane degradation in this organism, two putative genes for alkane hydroxylases were cloned from Alcanivorax borkumensis SK2. They were named alkB1 and alkB2. These genes were subsequently disrupted in A. borkumensis SK2, and the growth phenotypes of the disruptants were examined. The results indicate that the alkB1 gene is responsible for the degradation of short-chain n-alkanes. A double mutant defective in both alkB1 and alkB2 was still able to grow on medium-chain n-alkanes, indicating that genes other than alkB1 and alkB2 are also involved in n-alkane hydroxylation by A. borkumensis SK2.     

Differential protein expression during growth on linear versus branched alkanes in the obligate marine hydrocarbon-degrading bacterium Alcanivorax borkumensis SK2T

Alcanivorax borkumensis SK2^T is an important obligate hydrocarbonoclastic bacterium (OHCB) that can dominate microbial communities following marine oil spills. It possesses the ability to degrade branched alkanes which provides it a competitive advantage over many other marine alkane degraders that can only degrade linear alkanes. We used LC–MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on linear (n-C₁₄) or branched (pristane) alkanes. During growth on n-C₁₄, A. borkumensis expressed a complete pathway for the terminal oxidation of n-alkanes to their corresponding acyl-CoA derivatives including AlkB and AlmA, two CYP153 cytochrome P450s, an alcohol dehydrogenase and an aldehyde dehydrogenase. In contrast, during growth on pristane, an alternative alkane degradation pathway was expressed including a different cytochrome P450, an alcohol oxidase and an alcohol dehydrogenase. A. borkumensis also expressed a different set of enzymes for β-oxidation of the resultant fatty acids depending on the growth substrate utilized. This study significantly enhances our understanding of the fundamental physiology of A. borkumensis SK2^T by identifying the key enzymes expressed and involved in terminal oxidation of both linear and branched alkanes. It has also highlights the differential expression of sets of β-oxidation proteins to overcome steric hinderance from branched substrates.     

Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.     

Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2

Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents.     

Analysis of storage lipid accumulation in Alcanivorax borkumensis: Evidence for alternative triacylglycerol biosynthesis routes in bacteria

Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in A. borkumensis strain SK2. Triacylglycerols (TAGs) and wax esters (WEs), but not poly(hydroxyalkanoic acids), are the principal storage lipids present in this and other hydrocarbonoclastic bacterial species. Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. The acyltransferase AtfA1 (ABO_2742) exhibiting wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) activity plays a key role in both TAG and WE biosynthesis, whereas AtfA2 (ABO_1804) was dispensable for storage lipid formation. However, reduced but still substantial residual TAG levels in atfA1 and atfA2 knockout mutants compellingly indicate the existence of a yet unknown WS/DGAT-independent alternative TAG biosynthesis route. Storage lipids of A. borkumensis were enriched in saturated fatty acids and accumulated as insoluble intracytoplasmic inclusions exhibiting great structural variety. Storage lipid accumulation provided only a slight growth advantage during short-term starvation periods but was not required for maintaining viability and long-term persistence during extended starvation phases.     

Properties of Mutants of Bacteria Belonging the Genus Erwinia Devoid of Common Components of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Biochemical consequences of mutational damage to common components of the Erwinia phosphoenolpyruvate-dependent phosphotransferase system (the HPr protein and enzyme I) were studied. The transport of glucose, mannose, fructose, and mannitol inErwinia was shown to require a preliminary induction of proteins of the phosphotransferase system. A drastic decrease in the rate of the transport of these carbohydrates was observed in ptsI and ptsH mutants. A disturbance in the common components suppresses the synthesis of inducible enzymes (-galactosidase, complexes of pectolate lyases and cellulases) and renders it resistant to catabolite repression by glucose, but mutants were shown to retain intracellular cAMP content. Erwinia mutants devoid of common components of the system lack phytopathogenic features. The appearance of an intact ptsI allele in the cell completely repaired pleiotropic disturbances in these mutants.     

Taxonomy of Marine Bacteria: the Genus Beneckea

One-hundred-and-forty-five isolates of marine origin were submitted to an extensive physiological, nutritional, and morphological characterization. All strains were gram-negative, facultatively anaerobic, straight or curved rods which were motile by means of flagella. Glucose was fermented with the production of acid but no gas. Sodium but no organic growth factors were required. None of the strains were able to denitrify or fix molecular nitrogen. The results of nutritional and physiological tests were submitted to a numerical analysis. On the basis of phenotypic similarity, nine groups were established. These groups could be distinguished from one another by multiple, unrelated, phenotypic traits. Six groups which had deoxyribonucleic acid (DNA) containing 45 to 48 moles per cent guanine plus cytosine (GC) were assigned to a redefined genus Beneckea. All of the strains in this genus, when grown in liquid medium, had a single, polar flagellum. When grown on a solid medium, many strains had peritrichous flagella. Two groups were similar to previously described species and were designated B. alginolytica and B. natriegens. The remaining four groups were designated B. campbellii, B. neptuna, B. nereida, and B. pelagia. An additional group of phenotypically similar strains having the properties of the genus Beneckea was not included in the numerical analysis. These strains were readily separable from species of this genus and were designated B. parahaemolytica. Of the remaining groups, one was identified as Photobacterium fischeri. The other group (B-2) which had about 41 moles% GC content in its DNA could not be placed into existing genera.     

AtMGT9基因T-DNA插入突变体表型分析

利用功能互补,对拟南芥AtMGT9基因的Mg2＋转运功能进行了研究.同时对AtMGT9的两个T-DNA插入突变体株系（mgt9-1,mgt9-2）进行表型分析,mgt9-1只能得到杂合体植株,有花粉败育现象,mgt9-2有纯合体,但没有可见表型.基因序列分析表明,mgt9-1中T-DNA插入位点在起始密码子后,mgt9-2中T-DNA插入位点在靠近C端第2个跨膜区中间位置.功能互补和63Ni2＋示踪实验证明截短的mgt9-2蛋白能够互补细菌镁离子转运突变体MM281, 具有全序列基因一样的转运特性,而mgt9-1不具有转运功能,是一个功能缺失突变体.这为进一步研究AtMGT9蛋白的结构与功能的关系奠定了基础.     

Isolation and Genetic Analysis of Caulobacter Mutants Defective in Cell Shape and Membrane Lipid Synthesis

In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. One of these, a fatty acid bradytroph, AE6002, was shown to be due to a mutation in the fatA gene. In addition to the presence of the fatA506 mutation, this strain was found to contain two other mutations, one of which caused the production of a water-soluble brown-orange pigment (pigA) and another which caused formation of helical cells (hclA). Expression of the latter two phenotypes required complex media and both were repressed by glucose. However, the lesions were mapped to loci that are separated by a substantial distance. The hclA and the fatA genes mapped close together, possibly implying that comutation had occurred in AE6002. Data are presented that allow the unambiguous identification of a second Fat gene (fatB) in C. crescentus. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO₄ auxotroph gpsA505, was also determined. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location.     

Ail and PagC-Related Proteins in the Entomopathogenic Bacteria of Photorhabdus Genus

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1 _Pl and ail2 _Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1 _Pl expression was directly controlled by PhoPQ and low external Mg²⁺ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2 _Pl and pagC _Pl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.     

Mutations in the lux Operon of Natural Dark Mutants in the Genus Vibrio

Bacterial bioluminescence can display a wide range of intensities among strains, from very bright to undetectable, and it has been shown previously that there are nonluminous vibrios that possess lux genes. In this paper, we report the isolation and characterization of completely dark natural mutants in the genus Vibrio. Screening of over 600 Vibrio isolates with a luxA gene probe revealed that approximately 5% carried the luxA gene. Bioluminescence assays of the luxA-positive isolates, followed by repetitive extragenic palindromic-PCR fingerprinting, showed three unique genotypes that are completely dark. The dark mutants show a variety of lesions, including an insertion sequence, point mutations, and deletions. Strain BCB451 has an IS10 insertion sequence in luxA, a mutated luxE stop codon, and a truncated luxH. Strain BCB494 has a 396-bp deletion in luxC, and strain BCB440 has a frameshift in luxC. This paper represents the first molecular characterization of natural dark mutants and the first demonstration of incomplete lux operons in natural isolates.     

Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Identification and Genetic Characterization of Mutants of Bacteriophage T4 Defective in the Ability to Induce Exonuclease A

A mutant of bacteriophage T4 unable to induce exonuclease A has been isolated. The mutation responsible for this defect maps between genes 39 and 56, in a region of the chromosome devoid of other known markers. Four deletion mutants lacking part of the genome located between genes 39 and 56 also fail to induce exonuclease A. The ability of all of these mutants to replicate suggests that exonuclease A is not essential for replication of phage T4.     

Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, Wzy_Sf. Oag chain length is regulated by chromosomally encoded Wzz_Sf and pHS-2 plasmid-encoded Wzz_pHS2. To identify functionally important amino acid residues in Wzy_Sf, random mutagenesis was performed on the wzy_Sf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated Wzy_Sf proteins in the wzy_Sf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of Wzy_Sf and Wzz_Sf was investigated by transforming these mutated wzy_Sf plasmids into a wzy_Sf- and wzz_Sf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified Wzy_Sf mutants whose polymerization activities were Wzz_Sf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the Wzy_Sf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for Wzz_Sf in Wzy_Sf stability. Hence, in addition to its role in regulating Oag modal chain length, Wzz_Sf also affects Wzy_Sf activity and stability.     

Optimization of Multilocus Sequence Analysis for Identification of Species in the Genus Vibrio

Multilocus sequence analysis (MLSA) is an important method for identification of taxa that are not well differentiated by 16S rRNA gene sequences alone. In this procedure, concatenated sequences of selected genes are constructed and then analyzed. The effects that the number and the order of genes used in MLSA have on reconstruction of phylogenetic relationships were examined. The recA, rpoA, gapA, 16S rRNA gene, gyrB, and ftsZ sequences from 56 species of the genus Vibrio were used to construct molecular phylogenies, and these were evaluated individually and using various gene combinations. Phylogenies from two-gene sequences employing recA and rpoA in both possible gene orders were different. The addition of the gapA gene sequence, producing all six possible concatenated sequences, reduced the differences in phylogenies to degrees of statistical (bootstrap) support for some nodes. The overall statistical support for the phylogenetic tree, assayed on the basis of a reliability score (calculated from the number of nodes having bootstrap values of ≥80 divided by the total number of nodes) increased with increasing numbers of genes used, up to a maximum of four. No further improvement was observed from addition of the fifth gene sequence (ftsZ), and addition of the sixth gene (gyrB) resulted in lower proportions of strongly supported nodes. Reductions in the numbers of strongly supported nodes were also observed when maximum parsimony was employed for tree construction. Use of a small number of gene sequences in MLSA resulted in accurate identification of Vibrio species.     

Electrophoretic Analysis of Isoenzymes in the Identification of Trypanosomatids of the Genus Phytomonas1

Five strains of trypanosomatids of the genus Phytomonas, isolated from different species of Euphorbia {Euphorbia heterophylla, E. characias, E. pinea, E. hyssopifolia) and from Manihot escutenta, were cultured and compared through the electrophoretic mobility of isoenzymes of six enzymes: aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucosephosphate isomerase (EC 5.3.1.9), and malate dehydrogenase (EC 1.1.1.40). The strains could be distinguished from one another by their respective isoenzyme profiles.