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Archives of Microbiology (v.194, #4)

Regulation of Caenorhabditis elegans and Pseudomonas aeruginosa machinery during interactions by Balasubramanian Vigneshkumar; Shunmugiah Karutha Pandian; Krishnaswamy Balamurugan (pp. 229-242).

The amenability of Caenorhabditis elegans against pathogen provides a valuable tool for studying host–pathogen interactions. Physiological experiments revealed that the P. aeruginosa was able to kill C. elegans efficiently. The effects of P. aeruginosa PA14, PAO1 and their isolated lipopolysaccharide (LPS) on the host system were analyzed. The LPS at higher concentrations (≥2 mg/ml) was toxic to the host animals. Kinetic studies using qPCR revealed the regulation of host-specific candidate antimicrobial genes during pathogen-mediated infections. In addition, the pathogen-specific virulent gene, exoT expression, was anlyzed and found to be varied during the interactions with the host system. Ability of the pathogens to modify their internal machinery in the presence of the host was analyzed by XRD, FTIR and PCA. LPS isolated from pathogens upon exposure to C. elegans showed modifications at their functional regions. LPS from PAO1 showed difference in d-spacing angle (Å) and °2Th position. FTIR spectra revealed alterations in polysaccharide (1,200–900 cm⁻¹) and fatty acid (3,000–2,800 cm⁻¹) regions of LPS from P. aeruginosa PAO1 exposed to the host system. These data provide additional insights on how the pathogens subvert its own and host machinery during interactions.

Keywords: Caenorhabditis elegans ; Innate immunity; Lipopolysaccharide; Pseudomonas aeruginosa ; FTIR

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta by Masamichi Ide; Hirofumi Ichinose; Hiroyuki Wariishi (pp. 243-253).

We explored the molecular diversity and functional capabilities of cytochrome P450 monooxygenases (P450s) from the brown-rot basidiomycete Postia placenta. Using bioinformatic and experimental data, we found 250 genes of P450s in the whole genome, including 60 putative allelic variants. Phylogenetic analysis revealed the presence of 42 families, including 18 novel families. Comparative phylogenetic analysis of P450s from P. placenta and the white-rot basidiomycete Phanerochaete chrysosporium suggested that vigorous gene duplication and molecular evolution occurred after speciation of basidiomycetes. Among the 250 gene models, 184 were isolated as full-length cDNA and transformed into Saccharomyces cerevisiae to construct a functional library in which recombinant P450s were co-expressed with yeast NADPH-P450 oxidoreductase. Using this library, the catalytic potentials of P450s against a wide variety of compounds were investigated. A functionomic survey allowed the discovery of novel catalytic properties of P. placenta P450s. The phylogenetic diversity of the CYP53 family in P. placenta was clear, and CYP53D2 is capable of converting stilbene derivatives. This is the first report of this peculiar function of the CYP53 family. Our increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.

Keywords: Cytochrome P450; Brown-rot basidiomycete; Diversity; Functional screening; CYP53

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars by Magdalena Popowska; Magdalena Osińska; Magdalena Rzeczkowska (pp. 255-268).

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.

Keywords: Listeria monocytogenes ; N-acetylglucosamine-6-phosphate deacetylase; Amino-sugar catabolism; Peptidoglycan; Teichoic acid; Mutanolysin; Colistin

Expression of multiple tfb genes in different Halobacterium salinarum strains and interaction of TFB with transcriptional activator GvpE by Anne Bleiholder; Regina Frommherz; Katharina Teufel; Felicitas Pfeifer (pp. 269-279).

Halobacterium salinarum NRC-1 contains multiple TBP and TFB proteins required for the recruitment of RNA polymerase for transcription initiation. The presence and the expression of genes encoding TFB were investigated in the two Hbt. salinarum strains NRC-1 and PHH1 and the mutant strain PHH4. The plasmid-encoded tfbC and tfbE genes of NRC-1 were lacking in PHH1 and PHH4. The 5′-end of the tfbF transcript was determined and contained a 5′-untranslated region of 39 nucleotides able to form a stem-loop structure. The expression of these tfb genes was studied in cultures growing at 15, 37°C and under heat shock conditions. Cold temperatures reduced growth and except for tfbF also the amounts of all tfb transcripts. However, the formation of gas vesicles increased in PHH1 and NRC-1. Heat shock reduced growth of PHH1 and NRC-1, but PHH4 was not affected. A 100-fold increase in tfbA and tfbB mRNA was observed in PHH1 and PHH4, whereas NRC-1 reduced the amounts of these transcripts and increased the expression of tfbG. All TFB proteins tested were able to interact with the transcription activator GvpE involved in gas vesicle formation that thus is able to recruit TFB to the gvp promoter.

Keywords: TFB proteins; Genome variation; Alternative tfb genes

Identification of HrpX regulon genes in Xanthomonas oryzae pv. oryzicola using a GFP visualization technique by Yurong Li; Youlun Xiao; Lifang Zou; Huasong Zou; Gongyou Chen (pp. 281-291).

Xanthomonas oryzae pv. oryzicola is the causal agent of bacterial leaf streak in rice and injects repertoires of T3S effectors (T3SEs), which are normally regulated by a global regulator HrpX, into plant cells to suppress plant innate immunity for disease development. To establish a visualization technique to identify HrpX regulon genes in this pathogen, we chose six known or unknown T3SE genes of X. oryzae pv. oryzicola (strain RS105) as the targets in this report. The promoters of these candidates, whether or not containing a PIP-box, were fused with the gfp (green fluorescent protein) reporter gene, and the gfp reporters were introduced into the wild-type RS105 and the hrpX mutant RΔhrpX. GFP expression in the wild-type strain could be observed under fluorescence microscopy, but fluorescence was not observed in the hrpX mutant. Consequently, transcription and secretion detection demonstrated that these HrpX regulon members were repressed when hrpX was mutated and were not secreted into the hrp-inducing medium XOM3 because of a mutation in hrcV (T3SS deficient). These data suggest that the gfp reporter system is a feasible visualization tool for the identification of HrpX regulon genes and discovery of new T3SEs in the X. oryzae pv. oryzicola-rice pathosystem.

Keywords: X. oryzae pv. oryzicola ; HrpX regulon gene; gfp ; PIP-box

Transcriptional analysis and functional characterization of XCC1294 gene encoding a GGDEF domain protein in Xanthomonas campestris pv. campestris by Yi-Min Hsiao; Wan-Ling Song; Chao-Tsai Liao; I-Hsuan Lin; Mei-Ying Pan; Ching-Fen Lin (pp. 293-304).

The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.

Keywords: Cyclic AMP receptor protein-like protein; Reporter assay; Transcriptional regulation; Xanthomonas campestris pv. campestris

Global analysis of the Nitrosomonas europaea iron starvation stimulon by Neeraja Vajrala; Luis A. Sayavedra-Soto; Peter J. Bottomley; Daniel J. Arp (pp. 305-313).

The importance of iron to the metabolism of the ammonia-oxidizing bacterium Nitrosomonas europaea is well known. However, the mechanisms by which N. europaea acquires iron under iron limitation are less well known. To obtain insight into these mechanisms, transcriptional profiling of N. europaea was performed during growth under different iron availabilities. Of 2,355 N. europaea genes on DNA microarrays, transcripts for 247 genes were identified as differentially expressed when cells were grown under iron limitation compared to cells grown under iron-replete conditions. Genes with higher transcript levels in response to iron limitation included those with confirmed or assigned roles in iron acquisition. Genes with lower transcript levels included those encoding iron-containing proteins. Our analysis identified several potentially novel iron acquisition systems in N. europaea and provided support for the primary involvement of a TonB-dependent heme receptor gene in N. europaea iron homeostasis. We demonstrated that hemoglobin can act as an iron source under iron-depleted conditions for N. europaea. In addition, we identified a hypothetical protein carrying a lipocalin-like domain that may have the ability to chelate iron for growth in iron-limited media.

Keywords: Nitrosomonas europaea ; Fe; Iron limitation; Microarrays; Lipocalin; Hemoglobin

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Archives of Microbiology (v.194, #4)