Housekeeping genes essential for pantothenate biosynthesis are plasmid-encoded in <Emphasis Type="Italic">Rhizobium etli</Emphasis> and <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>

Background  

A traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of R. etli CFN42 predicts the presence of panC and panB genes clustered together on the 642 kb plasmid p42f and a second copy of panB on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the Rhizobiales with multipartite genomes, and assess the possibility that the panCB genes may have reached plasmids by horizontal gene transfer.     

Relationship between glucose catabolism and xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Two genes involved in central carbon metabolism were inactivated to modulate intracellular glucose 6-phosphate and to evaluate its effects on xanthan production in wild-type Xanthomonas oryzae pv. oryzae. Upon the inactivation of the phosphogluconate dehydratase gene (edd), intracellular glucose 6-phosphate increased from 0.05 to 1.17 mmol/g (dry cell wt). This was accompanied by increased xanthan production of up to 2.55 g/l (culture medium). In contrast, inactivation of 6-phosphogluconate dehydrogenase gene (gndA) did not influence intracellular glucose 6-phosphate nor xanthan production. The intracellular availability of glucose 6-phosphate is proposed as a rate-limiting factor in xanthan production, and it may be possible to increases production of xanthan by modulating the activities of enzymes in central carbon metabolism.     

Novel molecular markers of <Emphasis Type="Italic">Chlamydia pecorum</Emphasis> genetic diversity in the koala (<Emphasis Type="Italic">Phascolarctos cinereus</Emphasis>)

Background  

Chlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity.     

Identification of phenotypically and genotypically related <Emphasis Type="Italic">Lactobacillus</Emphasis> strains based on nucleotide sequence analysis of the <Emphasis Type="Italic">groEL,rpoB, rplB</Emphasis>, and <Emphasis Type="Italic">16S rRNA</Emphasis> genes

Sixty-eight cultures of lactic acid bacteria were isolated and identified from national fermented milk drinks (airan, koumiss, kurunga, shubat) home-made in different regions of the Republic of Kazakhstan and the Buryat Republic of Russia. The cultures of lactic acid bacteria of the genus Lactobacillus were identified as L. paracasei and L. rhamnosus related to the L. casei group and as L. brevis, L. buchneri, L. diolivorans, and L. parabuchneri (the L. buchneri group) using the classical microbiological methods and on the basis of the 16S rRNA gene sequence analysis. The polymorphism of the nucleotide sequences of the genes groEL, rpoB, and rplB encoding specific proteins was studied for intraspecific differentiation of the lactobacilli. The analysis of these genes allowed a more accurate identification of the lactobacilli that are genetically and phenotypically related to the L. casei group as L. paracasei subsp. paracasei and L. paracasei subsp. tolerans. The gene nucleotide sequences of all the genotyped strains were deposited in the GenBank database.     

Duplicate Gene Evolution Toward Multiple Fates at the <Emphasis Type="Italic">Drosophila melanogaster HIP</Emphasis>/<Emphasis Type="Italic">HIP-Replacement</Emphasis> Locus

Hsc/Hsp70-interacting protein (HIP) is a rapidly evolving Hsp70 cofactor. Analyses of multiple Drosophila species indicate that the HIP gene is duplicated only in D. melanogaster. The HIP region, in fact, contains seven distinctly evolving duplicated genes. The regional duplication occurred in two steps, fixed rapidly, and illustrates multiple modes of duplicate gene evolution. HIP and its duplicate HIP-R are adaptively evolving in a manner unique to the region: they exhibit elevated divergence from other drosophilids and low polymorphism within D. melanogaster. HIP and HIP-R are virtually identical, share polymorphisms, and are subject to gene conversion. In contrast, two other duplicate genes in the region, CG33221 and GP-CG32779, are pseudogenes, and the chimeric gene Crg1 is subject to balancing selection. HIP and HIP-R are evolving rapidly and adaptively; however, positive selection is not sufficient to explain the molecular evolution of the region as a whole. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ethanol Metabolism in the Yeasts <Emphasis Type="Italic">Yarrowia</Emphasis> and <Emphasis Type="Italic">Torulopsis</Emphasis>: A Review

Results of research into ethanol metabolism in yeast organisms with highly pronounced aerobic metabolism are reviewed. The low activity of NAD-dependent alcohol dehydrogenase (EC 1.1.1.1), observed under conditions of aerobic yeast growth on ethanol, demonstrates that alternative enzyme systems—alcohol oxidase (EC 1.1.3.13), microsomal ethanol-oxidizing system (including cytochrome P-450), and catalase (EC 1.11.1.6)—may be involved in the alcohol oxidation. The role of these systems in alcohol oxidation and the conditions favoring their operation in this processes are analyzed. It is concluded that iron ions are important regulators of ethanol metabolism for the microorganisms of this group.     

Overexpression of the Chitosanase Gene in <Emphasis Type="Italic">Fusarium solani</Emphasis> via <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

The <Emphasis Type="Italic">Nramp1</Emphasis>AA genotype confers susceptibility to <Emphasis Type="Italic">Brucella abortus</Emphasis> in water buffalo

The 3′ untranslated region of the water buffalo Nramp1 (natural resistance-associated macrophage protein 1) gene contains two alleles (Nramp1A and Nramp1B), as detected by the denaturing gradient gel electrophoresis (DGGE) technique. The Nramp1BB genotype is associated with resistance of water buffalo to the intracellular pathogen Brucella abortus. This article provides evidence that the Nramp1AA genotype is associated with susceptibility to the same pathogen. Susceptibility or resistance of water buffalo to B. abortus was established by agglutination, complement fixation, and skin tests. The Nramp1 genotype was established by DGGE analysis. The association between the Nramp1AA genotype and susceptibility to B. abortus was demonstrated in two independent population samples (152 cases and 281 controls; 87 cases and 124 controls, respectively). Macrophages from Nramp1AA subjects displayed a lower Nramp1 mRNA level when compared with macrophages from Nramp1BB subjects. Also, monocytes and macrophages from Nramp1AA subjects displayed a higher number of viable intracellular bacteria in comparison with monocytes and macrophages from Nramp1BB animals, providing biological significance to the results from association studies.     

Polygenic control for fermentation of β-fructosides in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: New genes <Emphasis Type="Italic">SUC9</Emphasis> and <Emphasis Type="Italic">SUC10</Emphasis>

Using molecular karyotyping and genetic hybridization analysis, two new polymeric β-fructosidase genes, SUC9 and SUC10, were identified in the yeast Saccharomyces cerevisiae, which are located on chromosome XIV and on the chromosome XVI/XIII doublet, respectively. The genes are responsible for fermentation of sucrose and raffinose. The SUC gene genotypes of strains VKM Y-1831 and DBVPG 1340 are SUC2 SUC9 and suc2 ⁰ SUC10, respectively. suc2 ⁰ is a silent sequence. The scientific and applied significance of SUC genes is discussed.     

<Emphasis Type="Italic">para</Emphasis>-Nitrophenol 4-monooxygenase and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-nitrocatechol in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. PnpA (PNP 4-monooxygenase) and PnpB (para-benzoquinone reductase) were shown to be involved in the initial steps of PNP catabolism via hydroquinone. We demonstrated here that PnpA also catalyzed monooxygenation of 4-nitrocatechol (4-NC) to hydroxyquinol, probably via hydroxyquinone. It was the first time that a single-component PNP monooxygenase has been shown to catalyze this conversion. PnpG encoded by a gene located in the PNP degradation cluster was purified as a His-tagged protein and identified as a hydroxyquinol dioxygenase catalyzing a ring-cleavage reaction of hydroxyquinol. Although all the genes necessary for 4-NC metabolism seemed to be present in the PNP degradation cluster in strain WBC-3, it was unable to grow on 4-NC as a sole source of carbon, nitrogen and energy. This was apparently due to the substrate’s inability to trigger the expression of genes involved in degradation. Nevertheless, strain WBC-3 could completely degrade both PNP and 4-NC when PNP was used as the inducer, demonstrating its potential in bioremediation of the environment polluted by both 4-NC and PNP.     

Interaction of <Emphasis Type="Italic">Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Complex regulation and multiple developmental functions of <Emphasis Type="Italic">misfire</Emphasis>, the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>ferlin gene

Background  

Ferlins are membrane proteins with multiple C2 domains and proposed functions in Ca²⁺ mediated membrane-membrane interactions in animals. Caenorhabditis elegans has two ferlin genes, one of which is required for sperm function. Mammals have several ferlin genes and mutations in the human dysferlin (DYSF) and otoferlin (OTOF) genes result in muscular dystrophy and hearing loss, respectively. Drosophila melanogaster has a single ferlin gene called misfire (mfr). A previous study showed that a mfr mutation caused male sterility because of defects in fertilization. Here we analyze the expression and structure of the mfr gene and the consequences of multiple mutations to better understand the developmental function of ferlins.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

<Emphasis Type="Italic">IXR1</Emphasis> and <Emphasis Type="Italic">HMO1</Emphasis> genes jointly control the level of spontaneous mutagenesis in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, γ-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.     

Quorum sensing regulation in <Emphasis Type="Italic">Pseudomonas</Emphasis>

Expression of many bacterial genes is regulated in a cell density-dependent manner via small signal molecules known as autoinducers; this type of regulation is termed quorum sensing (QS). The QS systems that employ N-acyl-homoserine lactones (HSLs) are best un derstood in Gram-negative bacteria. QS regulates expression of various genes, including the genes responsible for the production of virulence factors, synthesis of exoenzymes and antibiotics, antagonistic properties of bacteria, etc. The QS systems of the genus Pseudomonas are linked to other global regulatory networks of the cell, and their functions are controlled by numerous additional regulatory factors. Such regulators and the QS systems together form an intricate multifactorial cascade regulatory network. The review considers the QS systems of several Pseudomonas species, their interaction with other regulatory systems, and their roles in the regulation of cell processes.     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.