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Archives of Microbiology (v.189, #4)

New plasmid-mediated resistances to antimicrobial agents by Patrice Courvalin (pp. 289-291).

Stress responses in yeasts: what rules apply? by Pilar González-Párraga; Ruth Sánchez-Fresneda; María Martínez-Esparza; Juan-Carlos Argüelles (pp. 293-296).

Living organisms have evolved a complex network of mechanisms to face the unforeseen nutritional and environmental circumstances imposed on their natural habitats, commonly termed “stress”. To learn more about these mechanisms, several challenges are usually applied in the laboratory, namely nutrient starvation, heat shock, dehydration, oxidative exposures, etc. Yeasts are chosen as convenient models for studying stress phenomena because of their simple cellular organization and the amenability to genetic analysis. A vast scientific literature has recently appeared on the defensive cellular responses to stress. However, this plethora of studies covers quite different experimental conditions, making any conclusions open to dispute. In fact, the term “yeast stress” is rather confusing, since the same treatment may be very stressful or irrelevant, depending on the yeast. Customary expressions such as “gentle stress” (non-lethal) or “severe stress” (potentially lethal) should be precisely clarified. In turn, although prototypic yeasts share a common repertoire of signalling responsive pathways to stress, these are adapted to the specific ecological niche and biological activity of each particular species. What does “stress” really mean? Before we go any deeper, we have to define this uncertain meaning along with a proper explanation concerning the terms and conditions used in research on yeast stress.

Keywords: Yeast; Stress responses; Trehalose; Heat shock; Oxidative stress; Hog1; General response

The essential fatty acid myristate causes severe growth retardation in Hpelo disruptants of the yeast Hansenula polymorpha by Phatthanon Prasitchoke; Yoshinobu Kaneko; Takeshi Bamba; Ei-ichiro Fukusaki; Akio Kobayashi; Satoshi Harashima (pp. 297-304).

Myristate (C14:0) is an essential multi-functional fatty acid in a variety of organisms. We found that C14:0, but not other fatty acids, causes severe growth retardation in Hpelo1Δ and Hpelo2Δ mutants of the yeast Hansenula polymorpha, defective in elongation of very long-chain fatty acids. While transcription of HpELO1 and HpELO2 is transiently increased by C14:0, this was not found to be responsible for the growth retardation. Transcription of HpFAS1 and HpFAS2 encoding fatty acid synthase is repressed by C14:0, but this repression was also not found to be responsible for growth retardation. A screen for suppressors that resulted in restored growth of the Hpelo1Δ disruptant on media containing C14:0 identified two types of suppressors. One exhibited a defect in C14:0 uptake while the other did not. Molecular genetic and genomic analysis of these suppressor mutations is anticipated to shed new light on the processes of fatty acid transport and the crucial role of C14:0 in the growth of eukaryotic organisms.

Keywords: Myristate (C14:0); Growth retardation; Elongase for very long-chain fatty acids; HpELO1 ; HpELO2 ; Hansenula polymorpha ; Methylotrophic yeast; C₁-BODIPY-C₁₂

Iron and carbon metabolism by a mineral-oxidizing Alicyclobacillus-like bacterium by Adibah Yahya; Kevin B. Hallberg; D. Barrie Johnson (pp. 305-312).

A novel iron-oxidizing, moderately thermophilic, acidophilic bacterium (strain “GSM”) was isolated from mineral spoil taken from a gold mine in Montana. Biomolecular analysis showed that it was most closely related to Alicyclobacillus tolerans, although the two bacteria differed in some key respects, including the absence (in strain GSM) of ϖ-alicyclic fatty acids and in their chromosomal base compositions. Isolate GSM was able to grow in oxygen-free media using ferric iron as terminal electron acceptor confirming that it was a facultative anaerobe, a trait not previously described in Alicyclobacillus spp.. The acidophile used both organic and inorganic sources of energy and carbon, although growth and iron oxidation by isolate GSM was uncoupled in media that contained both fructose and ferrous iron. Fructose utilization suppressed iron oxidation, and oxidation of ferrous iron occurred only when fructose was depleted. In contrast, fructose catabolism was suppressed when bacteria were harvested while actively oxidizing iron, suggesting that both ferrous iron- and fructose-oxidation are inducible in this acidophile. Isolate GSM accelerated the oxidative dissolution of pyrite in liquid media either free of, or amended with, organic carbon, although redox potentials were significantly different in these media. The potential of this isolate for commercial mineral processing is discussed.

Keywords: Acidophile; Alicyclobacillus ; Firmicute ; Iron oxidation; Iron reduction; Mixotroph; Pyrite

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression by Dwayne A. Elias; Sandra L. Tollaksen; David W. Kennedy; Heather M. Mottaz; Carol S. Giometti; Jeffrey S. McLean; Eric A. Hill; Grigoriy E. Pinchuk; Mary S. Lipton; James K. Fredrickson; Yuri A. Gorby (pp. 313-324).

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.

Keywords: Controlled cultivation; Systems biology; Proteomics; Shewanella

Genotypic and phenotypic analysis of strains assigned to the widespread cyanobacterial morphospecies Phormidium autumnale (Oscillatoriales) by Katarzyna A. Palinska; Jürgen Marquardt (pp. 325-335).

In this study, ten cyanobacterial strains assigned to the oscillatorian species Phormidium autumnale have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, and pigment content. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The strains were quite homogenous in their morphologic features. Their thylakoids showed a stacked or fascicular pattern. Some, but not all strains contained phycoerythrin. Only one strain (P. autumnale UTCC 476) deviated significantly in its phenotype by lacking a calyptra. In neighbour-joining and maximum Parsimony trees most 16S rRNA sequences were located on a single well-defined branch, which, however, also harboured sequences assigned to other cyanobacterial genera. Two strains (P. autumnale UTCC 476 and P. autumnale UTEX 1580) were found on distant branches. The presence of phycoerythrin was not correlated with the strains’ position in the phylogenetic trees. Our results reconfirm that the morphospecies P. autumnale and the Phormidium group in general are not phylogenetically coherent and require revision. However, as indicated by sequence similarities most of the strains assigned to P. autumnale except P. autumnale UTCC 476 and P. autumnale UTEX 1580 are phylogenetically related and might belong to a single genus.

Keywords: Cyanobacteria; Oscillatoriales; Phormidium autumnale ; Phylogeny; Taxonomy

Occurrence and antagonistic potential of Stenotrophomonas strains isolated from deep-sea invertebrates by Lyudmila A. Romanenko; Masataka Uchino; Naoto Tanaka; Galina M. Frolova; Natalia N. Slinkina; Valery V. Mikhailov (pp. 337-344).

Stenotrophomonas maltophilia is known to be of significance as opportunistic pathogen as well as a source of biocontrol and bioremediation activities. S. maltophilia strains have been isolated from rhizospheres, soil, clinical material, aquatic habitats, but little is known about Stenotrophomonas strains recovered from marine environments. During a survey of the biodiversity of Pseudomonas-like bacteria associated with deep-sea invertebrates six Stenotrophomonas strains were isolated from sponge, sea urchin, and ophiura specimens collected from differing Pacific areas, including the Philippine Sea, the Fiji Sea and the Bering Sea. 16S rRNA gene sequence analysis confirmed an assignment of marine isolates to the genus Stenotrophomonas as it placed four strains into the S. maltophilia CIP 60.77^T cluster and two related to the S. rhizophila DSM 14405^T. Together with a number of common characteristics typical of S. maltophilia and S. rhizophila marine isolates exhibited differences in pigmentation, a NaCl tolerance, a range of temperatures, which supported their growth, substrate utilization pattern, and antibiotics resistance. Strains displayed hemolytic and remarkable inhibitory activity against a number of fungal cultures and Gram-positive microorganisms, but very weak or none against Candida albicans. This is the first report on isolation, taxonomic characterization and antimicrobial activity of Stenotrophomonas strains isolated from deep-sea invertebrates.

Keywords: Stenotrophomonas maltophilia ; Stenotrophomonas rhizophila ; Marine isolates; Phenotype; Phylogeny; Antifungal activity; Antibacterial activity; Deep-sea invertebrates

Signal molecules in the peanut–bradyrhizobia interaction by Tania Taurian; Belén Morón; María E. Soria-Díaz; Jorge G. Angelini; Pilar Tejero-Mateo; Antonio Gil-Serrano; Manuel Megías; Adriana Fabra (pp. 345-356).

Main nodulation signal molecules in the peanut–bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed.

Keywords: Arachis hypogaea L.; Flavonoids; Nod factors; Rhizobial isolates; Nodulation and fixation genes

Excision of the nifD element in the heterocystous cyanobacteria by B. J. Henson; L. E. Pennington; L. E. Watson; S. R. Barnum (pp. 357-366).

Heterocyst differentiation in cyanobacteria is accompanied by developmentally regulated DNA rearrangements that occur within the nifD, fdxN, and hupL genes. These genetic elements are excised from the genome by site-specific recombination during the latter stages of differentiation. The nifD element is excised by the recombinase, XisA, located within the element. Our objective was to examine the XisA-mediated excision of the nifD element. To accomplish this, we observed the ability of XisA to excise substrate plasmids that contained the flanking regions of the nifD element in an E. coli host. Using PCR directed mutagenesis, nucleotides in the nifD element flanking regions in substrate plasmids were altered and the effect on recombination was determined. Results indicate that only certain nucleotides within and surrounding the direct repeats are involved in excision. In some nucleotide positions, the presence of a purine versus a pyrimidine greatly affected recombination. Our results also indicated that the site of excision and branch migration occurs in a 6 bp region within the direct repeats.

Keywords: nifD Element; xisA ; Developmentally regulated DNA rearrangement; Site-specific recombination

Identification by gene deletion analysis of barS2, a gene involved in the biosynthesis of γ-butyrolactone autoregulator in Streptomyces virginiae by Yong Jik Lee; Shigeru Kitani; Hiroshi Kinoshita; Takuya Nihira (pp. 367-374).

Virginiae butanolide (VB) is a member of the γ-butyrolactone autoregulators and triggers the production of streptogramin antibiotics virginiamycin M₁ and S in Streptomyces virginiae. A VB biosynthetic gene (barS2) was localized in a 10-kb regulatory island which controls the virginiamycin biosynthesis/resistance of S. virginiae, and analyzed by gene disruption/complementation. The barS2 gene is flanked by barS1, another VB biosynthetic gene catalyzing stereospecific reduction of an A-factor-type precursor into a VB-type compound, and barX encoding a pleiotropic regulator for virginiamycin biosynthesis. The deduced product of barS2 possessed moderate similarity to a putative dehydrogenase of Streptomyces venezuelae, encoded by jadW 2 located in similar gene arrangement to that in the regulatory island of S. virginiae. A barS2-disruptant (strain IC152), created by means of homologous recombination, showed no differences in growth in liquid medium or morphology on solid medium compared to a wild-type strain, suggesting that BarS2 does not play any role in primary metabolism or morphological differentiation of S. virginiae. In contrast, no initiation of virginiamycin production or VB production was detected with the strain IC152 until 18 h of cultivation, at which time full production of virginiamycin occurs in the wild-type strain. The delayed virginiamycin production of the strain IC152 was fully restored to the level of the wild-type strain either by the exogenous addition of VB or by complementation of the intact barS2 gene, indicating that the lack of VB production at the initiation phase of virginiamycin production is the sole reason for the defect of virginiamycin production, and the barS2 gene is of primary importance for VB biosynthesis in S. virginiae.

Keywords: Streptomyces virginiae ; Butyrolactone autoregulator; Biosynthesis

Cloning and expression of trypanothione reductase from a New World Leishmania species by Denise Barçante Castro-Pinto; Marcelo Genestra; Gustavo B. Menezes; Mariana Waghabi; Antonio Gonçalves; Catarina De Nigris Del Cistia; Carlos Mauricio R. Sant’Anna; Leonor L. Leon; Leila Mendonça-Lima (pp. 375-384).

Trypanothione disulfide (T[S]₂), an unusual form of glutathione found in parasitic protozoa, plays a crucial role in the regulation of the intracellular thiol redox balance and in the defense against oxidative stress. Trypanothione reductase (TR) is central to the thiol metabolism in all trypanosomatids, including the human pathogens Trypanosoma cruzi, Trypanosoma brucei and Leishmania. Here we report the cloning, sequencing and expression of the TR encoding gene from L. (L.) amazonensis. Multiple protein sequence alignment of all known trypanosomatid TRs highlights the high degree of conservation and illustrates the phylogenetic relationships. A 3D homology model for L. amazonensis TR was constructed based on the previously reported Crithidia fasciculata structure. The purified recombinant TR shows enzyme activity and in vivo expression of the native enzyme could be detected in infective promastigotes, both by Western blotting and by immunofluorescence.

Keywords: Trypanothione reductase (EC 1.8.1.12); Leishmania (L.) amazonensis ; Thiol metabolism; Gene cloning; Leishmaniasis

Transcriptional activity of Pseudomonas aeruginosa fhp promoter is dependent on two regulators in addition to FhpR by Taija Koskenkorva; Niina Aro-Kärkkäinen; Daniel Bachmann; Hiroyuki Arai; Alexander D. Frey; Pauli T. Kallio (pp. 385-396).

The regulation of flavohemoglobin expression is complex and depending on its host organism requires a wide variety of different transcriptional regulators. In Pseudomonas aeruginosa, the flavohemoglobin (Fhp) and its cognate regulator FhpR form an NO-sensing and detoxifying system regulated by their common bidirectional promoter P _fhp/P _fhpR. The intergenic fhp–fhpR region of P. aeruginosa PAO1 was used as a bait to isolate proteins affecting the transcription of fhp and fhpR. In addition to the FhpR, we identified two previously uncharacterized P. aeruginosa proteins, PA0779 and PA3697. Both PA0779 and PA3697 were found to be essential for NO₃ ⁻ and NO₂ ⁻ induced P _fhp activity under aerobic and low-oxygen conditions, and needed for the full function of P _fhp/P _fhpR as NO responsive regulatory circuit under aerobic conditions. In addition, we show that the transcriptional activity of P _fhpR is highly inducible upon addition of SNP under aerobic conditions, but not by NO₃ ⁻, NO₂ ⁻ or under low-oxygen conditions, supporting the findings that FhpR is not the only factor affecting flavohemoglobin expression in P. aeruginosa.

Keywords: P . aeruginosa ; Flavohemoglobin; fhp ; Transcriptional regulation; fhpR ; PA0779; PA3697

Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant by Luc F. M. Rouws; Jean L. Simões-Araújo; Adriana S. Hemerly; José I. Baldani (pp. 397-405).

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5™Tnp Transposome™ (Epicentre). Up to 1 × 10⁶ mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.

Keywords: Nitrogen-fixing bacteria; Endophytes; Transposome; Inverse PCR; Flagella; Motility; Biofilm

Polyethylene glycol (PEG)–carboxylate–CoA synthetase is involved in PEG metabolism in Sphingopyxis macrogoltabida strain 103 by Akio Tani; Peechapack Somyoonsap; Toshiyuki Minami; Kazuhide Kimbara; Fusako Kawai (pp. 407-410).

Sphingopyxis macrogoltabida strain 103 possesses polyethylene-glycol (PEG)-inducible pegBCDAE operon encoding the genes relevant to PEG degradation. PEG is converted to PEG-carboxylate by PegA (PEG dehydrogenase) and PegC (PEG-aldehyde dehydrogenase). In this study, the recombinant PegE (homologous to acyl-CoA synthetases) was characterized. PegE was an acyl-CoA synthetase active for PEG-carboxylate and fatty acids. Judging from the nature of this kind of protein (located on the cytoplasmic membrane as a translocator), PegE might be responsible for the translocation of PEG-carboxylate from the periplasm into the cytoplasm or for the detoxification of strong acidity of the substrate.

Keywords: Polyethylene glycol metabolism; Acyl-CoA synthetase; Peg operon; Sphingopyxis macrogoltabida

Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp. by Radhey Shyam Sharma; Vandana Mishra; Asif Mohmmed; Cherukuri Raghavendra Babu (pp. 411-418).

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.

Keywords: Phage specificity; Lipopolysaccharide (LPS); Sesbania ; Sinorhizobium ; Rhizobium ; Azorhizobium caulinodans

S-Adenosylmethionine induces BldH and activates secondary metabolism by involving the TTA-codon control of bldH expression in Streptomyces lividans by Delin Xu; Hyung-Jin Kwon; Joo-Won Suh (pp. 419-426).

In the present study, a mechanism for S-adenosylmethionine (SAM) to promote secondary metabolism was characterized in terms of bldH sl expression in Streptomyces lividans. A previous study demonstrated that SAM, on application at 2 μM, induces the transcription of the strR promoter (strRp), which originates from Streptomyces griseus, in S. lividans. An inactivation study verified that bldH sl is essential to strRp transcription in S. lividans and it was demonstrated that the effects of SAM on the induction of strRp activity, on the transcription of redZ and actII-orf4, and on antibiotic production were compromised when the unique leucine TTA-codon of bldH sl was changed to TTG. Western blot analysis revealed that SAM supplementation enhances the expression of bldH sl when the TTA-codon was intact but not when the TTG replacement was provided. This study validates the involvement of BldH_sl in the potentiating effect of SAM on the antibiotic production and substantiates that SAM controls the expression of bldH sl through the TTA-codon control in translating bldH mRNA. It is argued here that the intracellular SAM-level modulates the maturation of bldA, which encodes the UUA-codon tRNA and controls secondary metabolism in S. lividans.

Keywords: S-adenosylmethionine; TTA-codon; bldH ; strR promoter; Antibiotic production; Streptomyces lividans

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Archives of Microbiology (v.189, #4)