Microbiology

PmrAB and PhoPQ Variants in Colistin-Resistant Enterobacter spp. Isolates in Korea Abstract In this study, we investigated the amino acid variations and mRNA expression of PhoPQ and PmrAB two-component regulatory systems in colistin-resistant Enterobacter cloacae isolates from Korea. We determined the nucleotide sequences of phoP, phoQ, pmrA, and pmrB in 51 colistin-resistant, 5 colistin-susceptible, and 8 skip-well isolates and consequently, the corresponding amino acid sequences as well. PhoPQ and PmrAB sequences showed large variations among the isolates (14, 67, 20, and 68 sites, respectively). Although there was some discrepancy between the genes, the colistin-resistant E. cloacae isolates were grouped into four clades and the susceptible isolates were grouped into two clades. We did not find any distinct amino acid substitutions associated with colistin resistance. Furthermore, mRNA expression of phoQ and pmrB was not significantly higher in the colistin-resistant isolates. Our data suggests that the colistin resistance mechanisms might be different in E. cloacae when compared to other gram-negative bacteria.

Oral Nickel Changes of Intestinal Microflora in Mice Abstract Nickel, a silver-colored metal found in nature, is a common cause of allergic contact dermatitis. Nevertheless, the existence of inflammatory reaction to oral nickel exposure remains controversial. The following study investigates whether oral nickel can change the intestinal microflora in mice. A total of 20 female ICR mice were randomly divided into two groups: the oral nickel group (Group O) and the control group (Group C). Group O received water containing 400 µM NiSO4·6H2O, while group C was given pure water for 21 days. The content of nickel in the kidneys was determined by atomic absorption spectrophotometry, while the composition of bacterial community in the cecum was detected by 16S rDNA sequencing. The results were subsequently validated by real-time quantitative PCR with genus and species specific primers. Compared to Group C, significantly higher nickel levels were observed in Group O (P = 0.016); however, our data suggested that oral administration of 400 µM NiSO4·6H2O was nontoxic to the animals. (No statistical difference in body animal weight was found between Group O and Group C, before and after oral administration of nickel.) At the genus level, significantly higher relative abundance of Bacteroides (P = 0.016) and Intestinimonas (P = 0.018), and significantly lower relative abundance of Lachnospiraceae_NK4A136_group (P = 0.002) and Lachnospiraceae_UCG-001_group (P = 0.042) were observed in the oral nickel group compared to the control group. In addition, Group O had significantly lower ratio of Firmicutes/Bacteroides (P = 0.008). The results of real-time quantitative PCR further confirmed that the amplicon mass of Bacteroides and B. fragilis in the Group O was significantly higher compared to C group (P = 0.034 and P = 0.02). Oral nickel could change the intestinal microflora in mice, thus suggesting that oral nickel alters the interaction between the host and the intestinal flora.

Functional Replacement of the BioC and BioH Proteins of Escherichia coli Biotin Precursor Biosynthesis by Ehrlichia chaffeensis Novel Proteins Abstract The biosynthesis of the pimelate moiety of biotin in Escherichia coli requires two specialized proteins, BioC and BioH. However, the enzymes that have BioC- or BioH-like activities show remarkable sequence diversity among biotin-producing bacteria. Here, we report that the intracellular rickettsial pathogen Ehrlichia chaffeensis encodes two novel proteins, BioT and BioU, which functionally replace the E. coli BioC and BioH proteins, respectively. The desthiobiotin assays demonstrated that these two proteins make pimeloyl-acyl carrier protein (ACP) from the substrate malonyl-ACP with the aid of the FAS II pathway, through the expected pimeloyl-ACP methyl ester intermediate. BioT and BioU homologues seem restricted to the species of Ehrlichia and its close relative, Anaplasma. Taken together, the synthesis of the biotin precursor in E. chaffeensis appears to be catalyzed by two novel BioC- and BioH-like proteins.

Association of Listeria monocytogenes LIPI-1 and LIPI-3 marker llsX with invasiveness Abstract Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. The evolution of its genome has exhibited differences in virulence among strains of the same species. Listeria monocytogenes LIPI-3 (Listeria Pathogenicity Island 3) and LIPI-1 (Listeria Pathogenicity Island 1) are considered responsible for the increased virulence in some strains. The aim of this study was to detect LIPI-1 genes and the llsX gene belonging to LIPI-3 in invasive strains of L. monocytogenes and to establish whether there is a relationship among the invasiveness, presence of the llsX and LIPI-1 genes, and the source of the strains. The results showed that 70% of the strains were invasive, and all these strains except one possessed LIPI-1, which suggests that although there is a correlation between LIPI-1 and invasiveness, the independent mechanisms of LIPI-1 may contribute to invasiveness. In contrast, 35% of the total strains were positive for llsX and were invasive; thus, the results revealed that there is a strong association between llsX and the invasiveness of L. monocytogenes in HEp-2 cells (HeLa contaminant/epithelial in origin). In addition, there is no other association with any other variable in this study. Moreover, the authors found that LIPI-1 and llsX are more frequently found in fresh than in frozen vegetables. Together, the findings provide an approximation for the better understanding of Listeriolysin S (LLS) and its role in the pathogenesis of L. monocytogenes, and a possible relation between virulence factors and food-storage temperature.

Insights into the Draft Genome Sequence of the Kiwifruit-Associated Pathogenic Isolate Pseudomonas fluorescens AHK-1 Abstract Pseudomonas fluorescens is a physiologically diverse species of bacteria present in many habitats, which possesses multifunctional traits that provide it with the capability to exhibit biological control activities, promote plant health or cause plant disease. Here, we present the draft genome sequence of the kiwifruit-associated pathogenic isolate AHK-1 of P. fluorescens, which was isolated from the diseased leaves of kiwifruit plants. The genome size of AHK-1 was found to be 7,035,786 bp, with a G + C content of 60.88%. It is predicted to contain a total of 6327 genes, of which 3998 were homologous to genes in the other two sequenced P. fluorescensisolates (SBW25 and GcM5-1A) and 946 were unique to AHK-1 based on comparative genomic analysis. Furthermore, we identified several candidate virulence factors in the genome of AHK-1, including the fliA gene encoding flagellar biosynthetic protein for biosynthesis, and the genes for components of type VI, III, and IV secretion systems. This genomic resource will serve as a reference for better understanding the genetics of pathogenic and non-pathogenic strains, and will help to elucidate the pathogenic mechanisms of P. fluorescens associated with plant disease.

Selection of Optimized Reference Genes for qRT-PCR Normalization in Xanthomonas campestris pv. campestris Cultured in Different Media Abstract Black rot is a cruciferous disease caused by Xanthomonas campestris pv. campestris (Xcc) and results in significant economic losses worldwide; therefore, elucidation of the mechanism of Xcc pathogenesis is urgently required. In this study, we aimed to select optimized reference genes to verify the relative quantification of virulent genes in Xcc. Xcc strains were cultured in three different media [basic medium (MMX), hrp-inducing medium (MMXC) and rich medium (NYG)] and the expression stability of five candidate genes [thymidylate synthase (thyA), DNA gyrase subunit B (gyrB), DNA-directed RNA polymerase subunit beta, glyceraldehyde-3-phosphate dehydrogenase and 16S ribosomal RNA (16S rRNA)] was evaluated using BestKeeper, GeNorm, and NormFinder software programs. Quantitative real-time PCR (qRT-PCR) analysis confirmed that two Xcc effector genes were hrpX/hrpG-regulated in MMXC using selected genes as controls. Finally, gyrB and thyA were validated as the optimized reference genes of Xcc cultured in MMXC, and qRT-PCR analysis was demonstrated to be an efficient alternative to Gus-activity detection for the analysis of Xcc expression. This information will be useful in the future studies of Xcc, especially those seeking new functional genes.

Identification and Characterization of an Autophagy-Related Gene Acatg12 in Acremonium chrysogenum Abstract Autophagy is a highly conserved mechanism to overcome various stresses and recycle cytoplasmic components and organelles. Ubiquitin-like (UBL) protein Atg12 is a key protein involved in autophagosome formation through stimulation of Atg8 conjugation to phosphatidylethanolamine. Here, we describe the identification of the autophagy-related gene Acatg12, encoding an Atg12 homologous protein in the cephalosporin C producing fungus Acremonium chrysogenum. Disruption of Acatg12 impaired the delivery and degradation of eGFP-Atg8, indicating that the autophagic process was blocked. Meanwhile, conidiation was dramatically reduced in the Acatg12 disruption mutant (∆Acatg12). In contrast, cephalosporin C production was increased twofold in ∆Acatg12, but fungal growth was reduced after 6 days fermentation. Consistent with these results, the transcriptional level of the cephalosporin biosynthetic genes was increased in ∆Acatg12. The results extend our understanding of autophagy in filamentous fungi.

Microbial Bioleaching of Ag, Au and Cu from Printed Circuit Boards of Mobile Phones Abstract Electronic waste (E-Waste) is consumed at high speed in the world. These residues contain metals that increase their price each year, generating new research on the ability of microorganisms to recover the metals from these wastes. Therefore, this work evaluated the biologic lixiviation of Cu, Ag and Au from printed circuit boards (PCB) of mobile phones by three strains of Aspergillus niger, Candida orthopsilosis, Sphingomonas sp. and their respective consortia, in addition to leaching with citric acid. The microorganisms were cultured in mineral media with 0.5 g of PCB, and the treatments with 1M citric acid were added the same amount of PCB. All treatments were incubated for 35 days at room temperature. The results showed that Sphingomonas sp. MXB8 and the consortium of C. orthopsilosis MXL20 and A. niger MXPE6 can increase their dry biomass by 147% and 126%, respectively, in the presence of PCB. In the bioleaching of metals, the inoculation of A. niger MXPE6, the consortium of Sphingomonas sp. MXB8/C. orthopsilosis MXL20 and Sphingomonas sp. MXB8 leached 54%, 44.2% and 35.8% of Ag. The consortium of A. niger MX5 and A. niger MXPE6 showed a leaching of 0.53% of Au. A. niger MX5 leaching 2.8% Cu. Citric acid increased Cu leaching by 280% compared to treatments inoculated with microorganisms. Although further research is required, A. niger MXPE6 and the consortium of Sphingomonas sp. MXB8/C. orthopsilosis MXL20 could be an alternative to recover Ag from PCB of mobile phones.

Phylogenetic Studies on the Prodigiosin Biosynthetic Operon Abstract Prodigiosin and undecylprodigiosin are tripyrrolic red pigmented antibiotics produced by certain bacteria. Many strains of Serratia and certain other Gammaproteobacteria produce prodigiosin and undecylprodigiosin is produced by certain strains of Streptomyces. This is a multistage process which involves the synthesis of a bipyrrolic compound from L-proline and its subsequent condensation with a mono pyrrole synthesized from 2-octenal in the case of prodigiosin and malonyl-CoA in the case of undecylprodigiosin respectively. We have carried out sequence analysis of the genes involved in the pathway and identified the distribution of the prodigiosin producing genes amongst the various bacteria which have been fully sequenced. The presence of the operon was clearly seen in certain clustered branches suggesting inheritance from a common ancestor. This was further confirmed by the absence of traits observed in horizontally acquired genes like, GC content variation, codon bias or the presence of mobile elements. Multiple sequence alignment of the promoter of the prodigiosin operon in seven fully sequenced Serratia marcescens strains showed excellent homology. Putative regulatory elements in this region were identified by sequence analysis studies and many of them have been found to influence pigment production. The undecylprodigiosin gene cluster on the other hand, shows homology to other gene clusters involved in the production of other pyrrole-containing antibiotics of the genus Streptomyces. This coupled with the presence of ORFs with three different promoters could indicate lateral gene transfer. Hence the evolution of undecylprodigiosin operon could be an example of convergent evolution.

Volatile Compounds Produced by Cyanobacteria Isolated from Mangrove Environment Abstract Cyanobacterial communities from the Brazilian Atlantic coast have been recently sampled through cultured and non-cultured approaches. The maintenance of cyanobacterial strains in laboratory cultures is an important source of material for biological and chemical evaluation as well as biotechnological investigations. In this way, this work aimed to identify, for the first time, by means of GC–MS analyses, the nonpolar chemical profiles of four morphologically distinct cyanobacterial strains: Cyanobium sp. CENA178, Cyanobium sp. CENA181, Oxynema sp. CENA135 and Nostoc sp. CENA175, which were previously isolated from Brazilian mangroves. Six distinct classes of volatile compounds were identified: acids, alcohols, fatty aldehydes, esters, ketones and aliphatic hydrocarbons, from which 12 compounds were detected. The predominant compounds were 1-octadecyne and tetradecanoic acid, obtained from Oxynema sp. CENA135 and; the last one being also observed in Cyanobium sp. CENA181. In addition, the aliphatic hydrocarbon heptadecane was produced by these cyanobacterial strains as well as by Nostoc sp. CENA175. The compounds produced by the studied cyanobacteria have already been reported as possessing pharmaceutical properties such as antioxidant, cytotoxic and antimicrobial activities, besides industrial importance as source of intermediates for biofuel production. It is also important to mention that, considering the number of non-identified compounds, which were not compatible with the searched databases, these strains are promising sources of new compounds, denoting the need for more studies. Accordingly, since these strains were isolated from saline or brackish waters, it is also expected that they might be cultivated in waters not used for human consumption, enabling a low-cost approach for biomass and metabolites production.