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Archives of Microbiology (v.186, #4)

Diverse endophytic bacteria isolated from a leguminous tree Conzattia multiflora grown in Mexico by En Tao Wang; Zhi Yuan Tan; Xian Wu Guo; Rolando Rodríguez-Duran; Gisela Boll; Esperanza Martínez-Romero (pp. 251-259).

Conzattia multiflora is a leguminous tree present only in Mexico and Guatemala. There is no record about its symbiotic or pathogenic microbes. In this study, we found that numerous bacteria with 10⁴–10⁶ individuals per gram of fresh epidermis were distributed in the tissue of this plant. All the bacteria isolated from the Conzattia epidermis were Gram-negative, facultative anaerobic rods and formed yellow or colorless colonies. They were identified as endophytes by inoculation tests. Some of the bacteria could significantly promote the growth of Conzattia seedlings. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes. The existence of plant-borne lineages of Salmonella indicates that the unexplored plants may harbor some unknown microbes.

Keywords: Diversity; Phylogeny; Endophytic bacteria; Conzattia ; Epidermis

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris by Abdul Matin; Seok Ryoul Jeong; Jane Faull; Antonio Ortega Rivas; Naveed Ahmed Khan (pp. 261-271).

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.

Keywords: Balamuthia mandrillaris ; Feeding; Brain endothelial cells; Bacteria; Encephalitis

The heat shock response in the cyanobacterium Synechocystis sp. Strain PCC 6803 and regulation of gene expression by HrcA and SigB by Abhay K. Singh; Tina C. Summerfield; Hong Li; Louis A. Sherman (pp. 273-286).

We report on the genome-wide response, based on DNA microarrays, of the cyanobacterium Synechocystis sp. PCC 6803 wild type and ΔsigB to a 15 min heat shock. Approximately 9% of the genes in wild type and ΔsigB were significantly regulated (P < 0.001) following this treatment, with chaperones induced the most. The absence of sigB had no dramatic effect on specific genes induced by heat shock, but did affect the level of transcription of the chaperones. In addition, sigE was induced in ΔsigB. Comparison of global gene expression of the wild type and the hrcA mutant at 30°C enabled us to examine the HrcA regulon, which included groESL and groEL2. Several genes belonging to specific functional groups (e.g., pilus biogenesis/assembly and phototaxis, biosynthesis of aromatic amino acids, murien sacculus and peptidoglycan, surface polysaccharides, and the Sec pathway) were differentially regulated following heat shock. We used results from knock-out mutants in sigB, sigD and sigE to construct a model of the network of group 2 sigma factor regulation upon each other. In this network, SigB represented the major node and SigE a secondary node. Overall, we determined that transcription of the heat-shock genes are regulated to various degrees by SigB, SigE and HrcA.

Keywords: Heat shock; Cyanobacteria; Sigma factors; HrcA; Microarrays; Differential gene expression; Chaperones

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes by Merike Merimaa; Eeva Heinaru; Merit Liivak; Eve Vedler; Ain Heinaru (pp. 287-296).

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.

Keywords: Phenol and p-cresol-degrading bacteria; Carbamoylphosphate synthase gene (carA); Phenol hydroxylase; Catechol 2,3-dioxygenase; pheBA operon

Cloning, expression and deletion of the cuticle-degrading protease BLG4 from nematophagous bacterium Brevibacillus laterosporus G4 by Baoyu Tian; Ning Li; Lihui Lian; Junwei Liu; Jinkui Yang; Ke-Qin Zhang (pp. 297-305).

Brevibacillus laterosporus G4, which was isolated from soil sample, kills free-living nematodes (Panagrellus redivius) and plant-parasite nematodes (Bursaphelenchus xylophilus) and degrades their cuticle in previous bioassay. Our works for B. laterosporus G4 had demonstrated that an extracellular alkaline protease BLG4 played a key role as a pathogenic factor in infection against nematode. In this study, the nematicidal activity of BLG4 was further verified by an in vitro assay with purified recombinant BLG4. The encoding gene of BLG4 was cloned and showed high degree of homology with the subtilisin subclass of serine protease gene and another reported cuticle-degrading protease gene from nematophagous bacterium Bacillus sp. B16. Deletion of BLG4 by homologous recombinant had a significant effect on the pathogenicity of B. laterosporus. In infection assays the BLG4-deficient strain (BLG4-6) lost about 50% of its nematocidal activity and in toxicity tests the mortality rate of nematodes decreased with ∼56% in comparison to wild-type strain. This is the first report analyzing the function of a subtilisin enzyme involved in bacterium against nematode at the molecular level, and it is possible to use B. laterosporus as a model to study host-parasite interaction and to gain detailed knowledge of the infection process.

Keywords: Pathogenic subtilisin; Host-parasite interaction; Gene deletion; Heterologous expression

Characterization of the type III export signal of the flagellar hook scaffolding protein FlgD of Escherichia coli by Corinna Weber-Sparenberg; Petra Pöplau; Heiner Brookman; Maike Rochón; Carolin Möckel; Monika Nietschke; Heinrich Jung (pp. 307-316).

Transport of flagellar structural proteins beyond the cytoplasmic membrane is accomplished by a type III secretory pathway [flagellar type III secretion system (fTTSS)]. The mechanism of substrate recognition by the fTTSS is still enigmatic. Using the hook scaffolding protein FlgD of Escherichia coli as a model substrate, it is demonstrated that the export signal is contained within the N-terminal 71 amino acids of FlgD. Analysis of frame-shift mutations and alterations of the nucleotide sequence suggest a proteinaceous nature of the signal. Furthermore, the physicochemical properties of the first about eight amino acids are crucial for export.

Keywords: Protein Export; Type III Protein Secretion; Flagella Assembly; FlgD

Production of piscicolin 126 by Carnobacterium maltaromaticum UAL26 is controlled by temperature and induction peptide concentration by Lucas J. Gursky; Nathaniel I. Martin; Darren J. Derksen; Marco J. van Belkum; Kamaljit Kaur; John C. Vederas; Michael E. Stiles; Lynn M. McMullen (pp. 317-325).

Carnobacterium maltaromaticum UAL26 produces the antimicrobial peptides (bacteriocins) piscicolin 126, first isolated from C. maltaromaticum JG126, and carnobacteriocin BM1, first isolated from C. maltaromaticum LV17. C. maltaromaticum UAL26 is especially inhibitory to strains of Listeria monocytogenes. Bacteriocin activity is not observable in the supernatant of cultures of UAL26 grown in liquid media at 25°C, but at temperatures less than 19°C bacteriocin activity can be detected. In contrast to JG126, the piscicolin 126 operon is downregulated in UAL26 at higher temperature, and piscicolin 126 mRNA is not detected when UAL26 is grown at 25°C. Bacteriocin production in UAL26 grown at 15°C can be induced by addition of 10⁻¹⁰ M of chemically synthesized piscicolin 126 induction peptide (PisN). However, induction of bacteriocin production in UAL26 grown at 25°C requires 10⁻⁷ M of PisN. The sequence of the piscicolin 126 operon in UAL26 contains 34 single nucleotide differences compared with the piscicolin 126 operon in JG126, including single nucleotide differences in the immunity, histidine kinase, dedicated ABC-transporter and accessory genes, as well as a single nucleotide deletion in the transport accessory gene. This deletion causes a frameshift, resulting in truncation of the PisE transport accessory protein in UAL26.

Cloning and characterization of a novel gene involved in nitrogen fixation in Heliobacterium chlorum: a possible regulatory gene by Jigjiddorj Enkh-Amgalan; Hiroko Kawasaki; Hirozo Oh-oka; Tatsuji Seki (pp. 327-337).

In the present study, the transcriptional properties of the nitrogen fixation gene cluster of Hbt. chlorum, a strictly anaerobic, gram-positive, phototrophic bacterium, were explored. The cluster consisted of eleven genes in the same orientation in the order nifI 1 , nifI 2 , nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB, and nifV as detected previously. An open reading frame (orf1) preceding these genes was revealed by further cloning. The orf1 was co-transcribed with downstream nif genes in a single polycistronic transcript, the transcription start site (TSS) was located upstream of the orf1, and a putative promoter was identified 10 bp preceding the TSS. Unlike most diazotrophs which have a σ⁵⁴-dependent −24/−12 promoter, the promoter was similar to the −35/−10 E. coli promoter. The orf1 had no nif homolog in DNA databases, and the highest level of identity (27% at amino acid level) was found with hutP, a positive regulatory gene of the histidine utilization (hut) operon in B. subtilis. Analogous to the regulatory mechanism of the hut operon in B. subtilis, it is conceivable that the orf1 product interacts with the terminator-like structure located downstream of the orf1 during N-deficient condition and prevents transcription termination; thus, the transcription continues into the nif structural genes.

Keywords: Heliobacterium ; Nitrogen fixation; Nif genes; Transcriptional terminator; Anti-termination; Real-time RT-PCR

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1 by M. Francisca Olmo-Mira; Purificación Cabello; Carmen Pino; Manuel Martínez-Luque; David J. Richardson; Francisco Castillo; M. Dolores Roldán; Conrado Moreno-Vivián (pp. 339-344).

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.

Keywords: NADH-nitrite reductase; Nitrate assimilation; Phototrophic bacteria; Siroheme

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Archives of Microbiology (v.186, #4)

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Archives of Microbiology (v.186, #4)