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      Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ 54 regulon

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          Abstract

          Background

          Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat.

          Results

          In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ 54-dependent manner. A more complete picture of the σ 54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ 54-dependent promoters, using a position weight matrix approach. One of these σ 54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ 54-dependent promoter.

          Conclusions

          Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ 54 regulon.

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          Most cited references44

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          Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.

          Y. H. Yang (2002)
          There are many sources of systematic variation in cDNA microarray experiments which affect the measured gene expression levels (e.g. differences in labeling efficiency between the two fluorescent dyes). The term normalization refers to the process of removing such variation. A constant adjustment is often used to force the distribution of the intensity log ratios to have a median of zero for each slide. However, such global normalization approaches are not adequate in situations where dye biases can depend on spot overall intensity and/or spatial location within the array. This article proposes normalization methods that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types of cDNA microarray experiments. The selection of appropriate controls for normalization is discussed and a novel set of controls (microarray sample pool, MSP) is introduced to aid in intensity-dependent normalization. Lastly, to allow for comparisons of expression levels across slides, a robust method based on maximum likelihood estimation is proposed to adjust for scale differences among slides.
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            Identifying DNA and protein patterns with statistically significant alignments of multiple sequences.

            Molecular biologists frequently can obtain interesting insight by aligning a set of related DNA, RNA or protein sequences. Such alignments can be used to determine either evolutionary or functional relationships. Our interest is in identifying functional relationships. Unless the sequences are very similar, it is necessary to have a specific strategy for measuring-or scoring-the relatedness of the aligned sequences. If the alignment is not known, one can be determined by finding an alignment that optimizes the scoring scheme. We describe four components to our approach for determining alignments of multiple sequences. First, we review a log-likelihood scoring scheme we call information content. Second, we describe two methods for estimating the P value of an individual information content score: (i) a method that combines a technique from large-deviation statistics with numerical calculations; (ii) a method that is exclusively numerical. Third, we describe how we count the number of possible alignments given the overall amount of sequence data. This count is multiplied by the P value to determine the expected frequency of an information content score and, thus, the statistical significance of the corresponding alignment. Statistical significance can be used to compare alignments having differing widths and containing differing numbers of sequences. Fourth, we describe a greedy algorithm for determining alignments of functionally related sequences. Finally, we test the accuracy of our P value calculations, and give an example of using our algorithm to identify binding sites for the Escherichia coli CRP protein. Programs were developed under the UNIX operating system and are available by anonymous ftp from ftp://beagle.colorado.edu/pub/consensus.
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              Nitrogen assimilation and global regulation in Escherichia coli.

              Nitrogen limitation in Escherichia coli controls the expression of about 100 genes of the nitrogen regulated (Ntr) response, including the ammonia-assimilating glutamine synthetase. Low intracellular glutamine controls the Ntr response through several regulators, whose activities are modulated by a variety of metabolites. Ntr proteins assimilate ammonia, scavenge nitrogen-containing compounds, and appear to integrate ammonia assimilation with other aspects of metabolism, such as polyamine metabolism and glutamate synthesis. The leucine-responsive regulatory protein (Lrp) controls the synthesis of glutamate synthase, which controls the Ntr response, presumably through its effect on intracellular glutamine. Some Ntr proteins inhibit the expression of some Lrp-activated genes. Guanosine tetraphosphate appears to control Lrp synthesis. In summary, a network of interacting global regulators that senses different aspects of metabolism integrates nitrogen assimilation with other metabolic processes.
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                Author and article information

                Journal
                BMC Microbiol
                BMC Microbiology
                BioMed Central
                1471-2180
                2010
                28 August 2010
                : 10
                : 231
                Affiliations
                [1 ]Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-000 São Paulo, SP, Brazil
                [2 ]Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-000 São Paulo, SP, Brazil
                [3 ]Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. dos Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brasil
                Article
                1471-2180-10-231
                10.1186/1471-2180-10-231
                3224663
                20799976
                188e96ab-94e9-47be-b9aa-23339cce69b9
                Copyright ©2010 da Silva Neto et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 April 2010
                : 28 August 2010
                Categories
                Research Article

                Microbiology & Virology
                Microbiology & Virology

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