Sulfolipid accumulation in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> disrupted in the <Emphasis Type="Italic">mce2</Emphasis> operon

Mycobacterium tuberculosis, the causative agent of tuberculosis, has a lipid-rich cell wall that serves as an effective barrier against drugs and toxic host cell products, which may contribute to the organism’s persistence in a host. M. tuberculosis contains four homologous operons called nice (mce1–4) that encode putative ABC transporters involved in lipid importation across the cell wall. Here, we analyzed the lipid composition of M. tuberculosis disrupted in the mce2 operon. High resolution mass spectrometric and thin layer chromatographic analyses of the mutant’s cell wall lipid extracts showed accumulation of SL-1 and SL₁₂₇₈ molecules. Radiographic quantitative analysis and densitometry revealed 2.9, 3.9 and 9.8-fold greater amount of [³⁵S] SL-1 in the mce2 operon mutant compared to the wild type M. tuberculosis during the early/mid logarithmic, late logarithmic and stationary phase of growth in liquid broth, respectively. The amount of [³⁵S] SL₁₂₇₈ in the mutant also increased progressively over the same growth phases. The expression of the mce2 operon genes in the wild type strain progressively increased from the logarithmic to the stationary phase of bacterial growth in vitro, which inversely correlated with the proportion of radiolabel incorporation into SL-1 and SL₁₂₇₈ at these phases. Since the mce2 operon is regulated in wild type M. tuberculosis, its cell wall may undergo changes in SL-1 and SL₁₂₇₈ contents during a natural course of infection and this may serve as an important adaptive strategy for M. tuberculosis to maintain persistence in a host.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

Gene <Emphasis Type="Italic">TAENIATA</Emphasis> Is a Novel Negative Regulator of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Homeobox Genes <Emphasis Type="Italic">KNAT1, KNAT2, KNAT6</Emphasis>, and <Emphasis Type="Italic">STM</Emphasis>

Mutant Arabidopsis thaliana taeniata (tae) plants are characterized by an altered morphology of leaves and the inflorescence. At the beginning of flowering, the inflorescence produces fertile flowers morphologically intermediate between a shoot and a flower. The recessive mutation tae also causes the formation of ectopic meristems and shoot rosettes on leaves. The expressivity of the mutant characters depend on the temperature and photoperiod. Analysis of the activity of KNOX class I genes in the leaves of the tae mutant has demonstrated the expression of genes KNAT2 and STM and an increase in the expression of genes KNAT1 and KNAT6 compared to wild-type leaves. These data indicate that the TAE gene negatively regulates the KNAT1, KNAT2, KNAT6, and STM genes.__________Translated from Genetika, Vol. 41, No. 8, 2005, pp. 1068–1074.Original Russian Text Copyright © 2005 by Lebedeva, Ezhova, Melzer.     

The mutation of a novel <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> SRL4 gene rescues the Lethality of <Emphasis Type="Italic">rad53</Emphasis> and <Emphasis Type="Italic">lcd1</Emphasis> mutations by modulating dNTP levels

The SRL4 (YPL033C) gene was initially identified by the screening of Saccharomyces cerevisiae genes that play a role in DNA metabolism and/or genome stability using the SOS system of Escherichia coli. In this study, we found that the srl4Delta mutant cells were resistant to the chemicals that inhibit nucleotide metabolism and evidenced higher dNTP levels than were observed in the wild-type cells in the presence of hydroxyurea. The mutant cells also showed a significantly faster growth rate and higher dNTP levels at low temperature (16 degrees C) than were observed in the wild-type cells, whereas we detected no differences in the growth rate at 30 degrees C. Furthermore, srl4Delta was shown to suppress the lethality of mutations of the essential S phase checkpoint genes, RAD53 and LCD1. These results indicate that SRL4 may be involved in the regulation of dNTP production by its function as a negative regulator of ribonucleotide reductase.     

Phytase activity and its regulation in a rhizospheric strain of <Emphasis Type="Italic">Serratia plymuthica</Emphasis>

Serratia plymuthica strain IC1270 isolated from the rhizosphere, possessing antagonistic activity towards a wide range of plant-pathogenic fungi, is able to hydrolyze phytate. Phytase activity was found intracellularly, while no activity was detected in the culture liquid. Optimum activity was found at pH 4-5; it completely disappeared at pH > 7.0 and 2.5. Phytase production was practically absent in the exponential phase and reached a maximum in the late stationary phase. Mutations of genes grrA and grrS, encoding GacA/GacS-like 2-component global regulatory system, or in gene rpoS encoding the sigma factor RpoS subunit of RNA polymerase, led to a deficiency in phytase production. Introduction into mutants of the respective wild-type genes cloned into the wide-range plasmid pJFF224-NX under the control of the bacteriophage T4 gene 32 promoter complemented this deficiency. This is the first report implicating the GacA/GacS global regulators and RpoS factor in phytase production in bacteria.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Differential composition of culture supernatants from wild-type <Emphasis Type="Italic">Brucella abortus</Emphasis> and its isogenic <Emphasis Type="Italic">virB</Emphasis> mutants

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis–trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.     

Extracellular DNA and Type IV pili mediate surface attachment by <Emphasis Type="Italic">Acidovorax</Emphasis><Emphasis Type="Italic">temperans</Emphasis>

Extracellular DNA can play a structural role in the microbial environment. Here evidence is presented that an environmental isolate of Acidovorax temperans utilises extracellular DNA for intercellular and cell-surface attachment and that Type IV pili and electrostatic interactions play a role in this interaction. Preliminary attempts to isolate and purify extracellular polysaccharides from A. temperans strain CB2 yielded significant amounts of DNA raising the question of whether this molecule was present as a structural component in the extracellular matrix. The role of DNA in attachment was indicated by experiments in which the addition of DNase to liquid medium inhibited the attachment of Acidovorax to glass wool. A Tn5 insertional mutant, lacking Type IV pili, was unable to initiate attachment. Addition of DNase caused rapid detachment of bound cells, but no detachment occurred when proteinase, RNase or inactivated DNase were used. Addition of MgCl₂ also caused significant detachment, supporting the possible mechanistic role of electrostatic interactions in the attachment process. Although attachment was apparent in early to mid-log phase growth, surprisingly DNA was not detected in the culture supernatant until late stationary phase and coincided with an appreciable loss of cell viability. This suggests that during log-phase growth attachment is mediated by eDNA that is released in low quantities and/or is highly localised within the extracellular matrix and also that stationary phase DNA release through widespread cell lysis may be a separate and unrelated event.     

The <Emphasis Type="Italic">pds2</Emphasis> mutation is a lesion in the <Emphasis Type="Italic">Arabidopsis</Emphasis> homogentisate solanesyltransferase gene involved in plastoquinone biosynthesis

Plastoquinone plays critical roles in photosynthesis, chlororespiration and carotenoid biosynthesis. The previously isolated pds2 mutant from Arabidopsis was deficient in tocopherol and plastoquinone accumulation, and the biochemical phenotype of this mutant could not be reversed by externally applied homogentisate, suggesting a later step in tocopherol and/or plastoquinone biosynthesis had been disrupted. Recently, the protein encoded by At3g11950 (AtHST) was shown to condense homogentisate with solanesyl diphosphate (SDP), the substrate for plastoquinone synthesis, but not phytyl diphosphate (PDP), the substrate for tocopherol biosynthesis. We have sequenced the AtHST allele in the pds2 mutant background and identified an in-frame 6 bp (2 aa) deletion in the gene. The pds2 mutation could be functionally complemented by constitutive expression of AtHST, demonstrating that the molecular basis for the pds2 mutation is this 6 bp-lesion in the AtHST gene. Confocal microscopy of EGFP tagged AtHST suggested that AtHST is localized to the chloroplast envelope, supporting the hypothesis that plastoquinone synthesis occurs in the plastid.     

<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.     

<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.