The Chemolithoautotroph Acidithiobacillus ferrooxidans Can Survive under Phosphate-Limiting Conditions by Expressing a C-P Lyase Operon That Allows It To Grow on Phosphonates

ABSTRACT The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans is of great importance in biomining operations. During the bioleaching of ores, microorganisms are subjected to a variety of environmental stresses and to the limitations of some nutrients, such as inorganic phosphate (Pi), which is an essential component for all living cells. Although the primary source of phosphorus for microorganisms is Pi, some bacteria are also able to metabolize Pi esters (with a C-O-P bond) and phosphonates (with a very inert C-P bond). By using bioinformatic analysis of genomic sequences of the type strain of A. ferrooxidans (ATCC 23270), we found that as part of a Pho regulon, this bacterium has a complete gene cluster encoding C-P lyase, which is the main bacterial enzyme involved in phosphonate (Pn) degradation in other microorganisms. A. ferrooxidans was able to grow in the presence of methyl-Pn or ethyl-Pn as an alternative phosphorus source. Under these growth conditions, a great reduction in inorganic polyphosphate (polyP) levels was seen compared with the level for cells grown in the presence of Pi. By means of reverse transcription-PCR (RT-PCR), DNA macroarrays, and real-time RT-PCR experiments, it was found that A. ferrooxidans phn genes were cotranscribed and their expression was induced when the microorganism was grown in methyl-Pn as the only phosphorus source. This is the first report of phosphonate utilization in a chemolithoautotrophic microorganism. The existence of a functional C-P lyase system is a clear advantage for the survival under Pi limitation, a condition that may greatly affect the bioleaching of ores.

Tricksy Business: Transcriptome Analysis Reveals the Involvement of Thioredoxin A in Redox Homeostasis, Oxidative Stress, Sulfur Metabolism, and Cellular Differentiation in Bacillus subtilis

ABSTRACT Thioredoxins are important thiol-reactive proteins. Most knowledge about this class of proteins is derived from proteome studies, and little is known about the global transcriptional response of cells to various thioredoxin levels. In Bacillus subtilis, thioredoxin A is encoded by trxA and is essential for viability. In this study, we report the effects of minimal induction of a strain carrying an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible trxA gene (ItrxA) on transcription levels, as determined by DNA macroarrays. The effective depletion of thioredoxin A leads to the induction of genes involved in the oxidative stress response (but not those dependent on PerR), phage-related functions, and sulfur utilization. Also, several stationary-phase processes, such as sporulation and competence, are affected. The majority of these phenotypes are rescued by a higher induction level of ItrxA, leading to an approximately wild-type level of thioredoxin A protein. A comparison with other studies shows that the effects of thioredoxin depletion are distinct from, but show some similarity to, oxidative stress and disulfide stress. Some of the transcriptional effects may be linked to thioredoxin-interacting proteins. Finally, thioredoxin-linked processes appear to be conserved between prokaryotes and eukaryotes.

Expression Profiling of Virulence and Pathogenicity Genes of Xanthomonas axonopodis pv. citri

ABSTRACT DNA macroarrays of 279 genes of Xanthomonas axonopodis pv. citri potentially associated with pathogenicity and virulence were used to compare the transcriptional alterations of this bacterium in response to two synthetic media. Data analysis indicated that 31 genes were up-regulated by synthetic medium XVM2, while only 7 genes were repressed. The results suggest that XVM2 could be used as an in vitro system to identify candidate genes involved in pathogenesis of X. axonopodis pv. citri.

Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray

ABSTRACT Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.

Global Analyses of Transcriptomes and Proteomes of a Parent Strain and an l-Threonine-Overproducing Mutant Strain

ABSTRACT We compared the transcriptome, proteome, and nucleotide sequences between the parent strain Escherichia coli W3110 and the l-threonine-overproducing mutant E. coli TF5015. DNA macroarrays were used to measure mRNA levels for all of the genes of E. coli, and two-dimensional gel electrophoresis was used to compare protein levels. It was observed that only 54 of 4,290 genes (1.3%) exhibited differential expression profiles. Typically, genes such as aceA, aceB, icdA, gltA, glnA, leu operon, proA, thrA, thrC, and yigJ, which are involved in the glyoxylate shunt, the tricarboxylic acid cycle, and amino acid biosynthesis (l-glutamine, l-leucine, proline, and l-threonine), were significantly upregulated, whereas the genes dadAX, hdeA, hdeB, ompF, oppA, oppB, oppF, yfiD, and many ribosomal protein genes were downregulated in TF5015 compared to W3110. The differential expression such as upregulation of thr operon and expression of yigJ would result in an accumulation of l-threonine in TF5015. Furthermore, two significant mutations, thrA345 and ilvA97, which are essential for overproduction of l-threonine, were identified in TF5015 by the sequence analysis. In particular, expression of the mutated thrABC (pATF92) in W3110 resulted in a significant incremental effect on l-threonine production. Upregulation of aceBA and downregulation of b1795, hdeAB, oppA, and yfiD seem to be linked to a low accumulation of acetate in TF5015. Such comprehensive analyses provide information regarding the regulatory mechanism of l-threonine production and the physiological consequences in the mutant stain.

Development of Sinorhizobium meliloti Pilot Macroarrays for Transcriptome Analysis

ABSTRACT In order to prepare for whole-genome expression analysis in Sinorhizobium meliloti, pilot DNA macroarrays were designed for 34 genes of known regulation. The experimental parameters assessed were the length of the PCR products, the influence of a tag at the 5′ end of the primers, and the method of RNA labeling. Variance and principal-component analysis showed that the most important nonbiological parameter was the labeling method. The sizes of PCR products were also found to be important, whereas the influence of 5′ tags was minimal. The variability between replicated spots on a membrane was found to be low. These experimental procedures were validated by analyzing the effects of microaerobic conditions on gene expression.