Regulation of nitrogen metabolism in gram-positive bacteria

A search for new members of the TnrA and GlnR regulons, responsible for assimilation of nitrogen in Gram-positive bacteria, was performed. Common regulatory signals with consensus sequences ATGTNAWWWWWWWTNACAT and TGTNAWWWWWWWTNACA were identified for GlnR and TnrA, respectively. The structure was described and new potential members were found in Bacillus subtilis, B. licheniformis, Geobacillus kaustophilus, and Oceanobacillus iheyensis for the TnrA/GlnR regulons; in B. halodurans for the TnrA regulon; and in Lactococcus lactis, Lactobacillus plantarum, Streptococcus pyogenes, S. pneumoniae, S. mutans, S. agalactiae, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, and St. epidermidis for the GlnR regulon.     

Does Streptococcus mitis,a neonatal oropharyngeal bacterium,influence the pathogenicity of Pseudomonas aeruginosa?

Microorganisms in a biofilm might promote or suppress each other. We previously found that Pseudomonas aeruginosa (P. aeruginosa) and the normal colonized bacteria in the oropharynx, Streptococcus mitis (S. mitis), were the most common bacteria in the biofilm found on newborns' endotracheal tubes. Here, we found that S. mitis enhanced the adhesion and biofilm formation of P. aeruginosa. Furthermore, it alleviated the immune response induced by P. aeruginosa. These findings remind us that we should not ignore the role of traditionally viewed non-pathogenic bacteria in biofilms and provide new insights into exploring bacterial interaction mechanisms in biofilm related infections.     

Diversity and metabolic potential of culturable bacteria from the rhizosphere of Turkish tea grown in acidic soils

The purpose of this study was to investigate the diversity of cultivable phosphate solubilising (PSB) and total bacteria originated from 384 rhizospheric acidic soils samples of tea plants grown at 32 locations. Over 900 rhizoplane bacteria were randomly selected from agar-solidified trypticase soy broth, and identified using fatty acid methyl ester (FAME) profiles. Based on FAME profiles, 53 bacterial genera were identified with a similarity index >0.3, but 60.3% of the identified isolates belonged to five genera: Bacillus (34.6%), Pseudomonas (8.9%), Stenotrophomonas (6.1%), Paenibacillus (5.9%) and Arthrobacter (4.8%). The bacilli group comprised many different species, with the most abundant being B. cereus, B. megaterium and B. sphaericus. The main identified Pseudomonads included P. fluorescens, P. putida, and P. alcaligenes. About 30.4% of the bacterial isolates could not be classified to genus since their similarity indices were <0.3 indicating no close matches. Most of the total and P-solubilizing bacteria isolated were Gram positive (61.3 and 52.3%), and Gram negative constituted only 38.7 and 47.7%. Out of the 214 PSB from a pool of 506 bacterial isolates recovered on the selective media from the rhizosphere of tea, 74 of them were characterized by carbon sources using BIOLOG^M GN2 and GP2 plates. Bacillus, Pseudomonas, Paenibacillus and Stenotrophomonas genera were the most prominent P-solubilizing groups in the rhizosphere and soil populations analyzed. B. cereus, P. fluorescens, S. maltophilia, B. megaterium, P. putida, B. sphaericus and Paenibacillus polymyxa were the most frequent P-solubilizing species in the acidic tea rhizosohere soils. Selected Gram-positive PSB appeared to favour carbohydrates, and Gram-negative bacteria appeared to favour carboxylic acids, amino acids and carbohydrates as carbon sources. Selected phosphate solubilizing acid tolerant strains showed high variability in utilizing various carbon sources.     

Production of Antimicrobial Substances by Bacteria Isolated from Fermented Table Olives

Summary The production of antimicrobial substances was studied among 195 bacterial isolates from retail table olives. A total 86 isolates tested positive, and they clustered in 10 groups according to their inhibitory spectra. Many isolates (38.37%) produced strong inhibition against all bacteria tested (Listeria innocua, Lactococcus lactis, Bacillus cereus, B. megaterium, Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis, and Escherichia coli). The selected bacterial isolates were Gram-positive bacteria with rod morphology (62.67%), short rods (26.65%) or cocci (10.67%). Isolates producing antimicrobial substances may be useful as starters to enhance control of table olive fermentation and improve the safety of retail table olives.     

Bacteria associated with Amblyomma cajennense tick eggs

Ticks represent a large group of pathogen vectors that blood feed on a diversity of hosts. In the Americas, the Ixodidae ticks Amblyomma cajennense are responsible for severe impact on livestock and public health. In the present work, we present the isolation and molecular identification of a group of culturable bacteria associated with A. cajennense eggs from females sampled in distinct geographical sites in southeastern Brazil. Additional comparative analysis of the culturable bacteria from Anocentor nitens, Rhipicephalus sanguineus and Ixodes scapularis tick eggs were also performed. 16S rRNA gene sequence analyses identified 17 different bacterial types identified as Serratia marcescens, Stenotrophomonas maltophilia, Pseudomonas fluorescens, Enterobacter spp., Micrococcus luteus, Ochrobactrum anthropi, Bacillus cereus and Staphylococcus spp., distributed in 12 phylogroups. Staphylococcus spp., especially S. sciuri, was the most prevalent bacteria associated with A. cajennense eggs, occurring in 65% of the samples and also frequently observed infecting A. nitens eggs. S. maltophilia, S. marcescens and B. cereus occurred infecting eggs derived from specific sampling sites, but in all cases rising almost as pure cultures from infected A. cajennense eggs. The potential role of these bacterial associations is discussed and they possibly represent new targets for biological control strategies of ticks and tick borne diseases.     

Study of gut bacterial diversity of Bombyx mandarina and Bombyx mori through 16S rRNA gene sequencing

The wild silkworm B. mandarina is living in the natural environment has a strong stress resistance and adaptability after harsh natural selection. The indoor rearing or domestication of the wild silkworm under artificial custody for long period deteriorates stress resistance and ecological adaptability. Therefore, we aimed to investigate the effects of artificial domestication and evolutionary pressure on the gut bacterial diversity of B. mandarina and B. mori. The intestinal content of 6th day of fifth instar B. mandarina and B. mori larvae were analyzed by sequencing of the 16S rRNA gene through Illumina miseq sequencing technology. The outcome of the study revealed that abundance of predominant bacteria of phylum Firmicutes were respectively 81.40% and 81.85% in the late fifth instar silkworm larvae (6th day) of B. mandarina and B. mori. In Firmicutes, abundance of predominant bacterial genus Enterococcus in B. mandarina (69.73%) was comparatively higher than B. mori (48.99%). The genus Advenella belongs to phylum Proteobacteria was recorded only in B. mandarina (11.54%). The abundance of Unclassified_Peptostreptococcaceae, Methanobrevibacter, Ignatzschineria, Petrimonas and Proteiniphilum in B. mandarina were between 0.12 and 0.17%, nevertheless, these bacterial genera were not detected in B. mori. The abundance of genera Lactococcus, Bacillus and Pseudomonas in B. mori (17.73%, 5.02%, and 1.61%) were remarkably higher than B. mandarina (0.15%, 0.54% and 0.45%). These results indicated that substantial difference was observed between the intestinal bacteria of B. mori and B. mandarina population, and structure of the intestinal bacteria could be affected by the artificial domestication and evolutionary pressure.     

Endophytic Colonization and In Planta Nitrogen Fixation by a Herbaspirillum sp. Isolated from Wild Rice Species

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and ¹⁵N₂ gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

Platelet and neutrophil responses to Gram positive pathogens in patients with bacteremic infection

Background

Many Gram-positive pathogens aggregate and activate platelets in vitro and this has been proposed to contribute to virulence. Platelets can also form complexes with neutrophils but little is however known about platelet and platelet-neutrophil responses in bacterial infection.

Methodology/Principal Findings

We added isolates of Gram-positive bacteria from 38 patients with a bacteremic infection to blood drawn from the same patient. Aggregometry and flow cytometry were used to assess platelet aggregation and to quantify activation of platelets, neutrophils, and platelet-neutrophils complexes (PNCs) induced by the bacteria. Fifteen healthy persons served as controls. Most isolates of Staphylococcus aureus, beta hemolytic streptococci, and Enterococcus faecalis induced aggregation of platelets from their respective hosts, whereas pneumococci failed to do so. S. aureus isolates induced platelet aggregation more rapidly in patients than in controls, whereas platelet activation by S. aureus was lower in patients than in controls. PNCs were more abundant in baseline samples from patients than in healthy controls and most bacterial isolates induced additional PNC formation and neutrophil activation.

Conclusion/Significance

We have demonstrated for the first time that bacteria isolated from patients with Gram-positive bacteremia can induce platelet activation and aggregation, PNC formation, and neutrophil activation in the same infected host. This underlines the significance of these interactions during infection, which could be a target for future therapies in sepsis.     

Universal primers for detection of common bacterial pathogens causing prosthetic joint infection

The diagnosis of low grade prosthetic joint infection is difficult and time consuming. Nested-PCR for universal bacterial DNA segments detection of "orthopaedic" bacteria was tested in a laboratory setting. This method is based on amplification of the 16S bacterial ribosomal RNA coding sequences. 11 species of the most frequent bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens) involved in prosthetic joint infections were studied. All could be detected rapidly and sensitively by this method.     

Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections

A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.     

Novel multispecies microbial consortia involved in lignocellulose and 5-hydroxymethylfurfural bioconversion

To develop a targeted metagenomics approach for the analysis of novel multispecies microbial consortia involved in the bioconversion of lignocellulose and furanic compounds, we applied replicated sequential batch aerobic enrichment cultures with either pretreated or untreated wheat straw as the sources of carbon and energy. After each transfer, exponential growth of bacteria was detected using microscopic cell counts, indicating that the substrate was being utilized. In batch, the final bacterial abundances increased from an estimated 5 to 8.7–9.5 log 16S rRNA gene copy numbers/ml. The abundances of fungal propagules showed greater variation, i.e., between 5.4 and 8.0 log ITS1 copies/ml. Denaturing gradient gel electrophoresis analyses showed that the bacterial consortia in both treatments reached approximate structural stability after six transfers. Moreover, the structures of the fungal communities were strongly influenced by substrate treatment. A total of 124 bacterial strains were isolated from the two types of enrichment cultures. The most abundant strains were affiliated with the genera Raoultella/Klebsiella, Kluyvera, Citrobacter, Enterobacter, Pseudomonas, Acinetobacter, Flavobacterium and Arthrobacter. Totals of 43 and 11 strains obtained from the untreated and pretreated substrates, respectively, showed (hemi)cellulolytic activity (CMC-ase and xylanase), whereas 96 strains were capable of growth in 7.5 mM 5-hydroxymethylfurfural. About 50 % of the latter showed extracellular oxidoreductase activity as detected by a novel iodide oxidation method. Also, (hemi)cellulolytic fungal strains related to Coniochaeta, Plectosphaerella and Penicillium were isolated. One Trichosporon strain was isolated from pretreated wheat straw. The two novel bacterial–fungal consortia are starting points for lignocellulose degradation applications.     

Genetic Diversity of Viable, Injured, and Dead Fecal Bacteria Assessed by Fluorescence-Activated Cell Sorting and 16S rRNA Gene Analysis

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.     

Newly Cultured Bacteria with Broad Diversity Isolated from Eight-Week Continuous Culture Enrichments of Cow Feces on Complex Polysaccharides

One of the functions of the mammalian large intestinal microbiota is the fermentation of plant cell wall components. In ruminant animals, the majority of their nutrients are obtained via pregastric fermentation; however, up to 20% can be recovered from microbial fermentation in the large intestine. Eight-week continuous culture enrichments of cattle feces with cellulose and xylan-pectin were used to isolate bacteria from this community. A total of 459 bacterial isolates were classified phylogenetically using 16S rRNA gene sequencing. Six phyla were represented: Firmicutes (51.9%), Bacteroidetes (30.9%), Proteobacteria (11.1%), Actinobacteria (3.5%), Synergistetes (1.5%), and Fusobacteria (1.1%). The majority of bacterial isolates had <98.5% identity to cultured bacteria with sequences in the Ribosomal Database Project and thus represent new species and/or genera. Within the Firmicutes isolates, most were classified in the families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Clostridiaceae I. The majority of the Bacteroidetes were most closely related to Bacteroides thetaiotaomicron, B. ovatus, and B. xylanisolvens and members of the Porphyromonadaceae family. Many of the Firmicutes and Bacteroidetes isolates were related to species demonstrated to possess enzymes which ferment plant cell wall components; the others were hypothesized to cross-feed these bacteria. The microbial communities that arose in these enrichment cultures had broad bacterial diversity. With over 98% of the isolates not represented as previously cultured, there are new opportunities to study the genomic and metabolic capacities of these members of the complex intestinal microbiota.     

Sulfur bacteria in wastewater stabilization ponds periodically affected by the ‘red-water’ phenomenon

Several wastewater stabilization ponds (WSP) in Tunisia suffer periodically from the ‘red-water’ phenomenon due to blooming of purple sulfur bacteria, indicating that sulfur cycle is one of the main element cycles in these ponds. In this study, we investigated the microbial diversity of the El Menzeh WSP and focused in particular on the different functional groups of sulfur bacteria. For this purpose, we used denaturing gradient gel electrophoresis of PCR-amplified fragments of the 16S rRNA gene and of different functional genes involved in microbial sulfur metabolism (dsrB, aprA, and pufM). Analyses of the 16S rRNA revealed a relatively high microbial diversity where Proteobacteria, Chlorobi, Bacteroidetes, and Cyanobacteria constitute the major bacterial groups. The dsrB and aprA gene analysis revealed the presence of deltaproteobacterial sulfate-reducing bacteria (i.e., Desulfobacter and Desulfobulbus), while the analysis of 16S rRNA, aprA, and pufM genes assigned the sulfur-oxidizing bacteria community to the photosynthetic representatives belonging to the Chlorobi (green sulfur bacteria) and the Proteobacteria (purple sulfur and non sulfur bacteria) phyla. These results point on the diversity of the metabolic processes within this wastewater plant and/or the availability of sulfate and diverse electron donors.     

MALDI-TOF Identification of the Human Gut Microbiome in People with and without Diarrhea in Senegal

Background

In Africa, there are several problems with the specific identification of bacteria. Recently, MALDI-TOF mass spectrometry has become a powerful tool for the routine microbial identification in many clinical laboratories.

Methodology/Principal Findings

This study was conducted using feces from 347 individuals (162 with diarrhea and 185 without diarrhea) sampled in health centers in Dakar, Senegal. Feces were transported from Dakar to Marseille, France, where they were cultured using different culture conditions. The isolated colonies were identified using MALDI-TOF. If a colony was unidentified, 16S rRNA sequencing was performed. Overall, 2,753 isolates were tested, allowing for the identification of 189 bacteria from 5 phyla, including 2 previously unknown species, 11 species not previously reported in the human gut, 10 species not previously reported in humans, and 3 fungi. 2,718 bacterial isolates (98.8%) out of 2,750 yielded an accurate identification using mass spectrometry, as did the 3 Candida albicans isolates. Thirty-two bacterial isolates not identified by MALDI-TOF (1.2%) were identified by sequencing, allowing for the identification of 2 new species. The number of bacterial species per fecal sample was significantly higher among patients without diarrhea (8.6±3) than in those with diarrhea (7.3±3.4; P = 0.0003). A modification of the gut microbiota was observed between the two groups. In individuals with diarrhea, major commensal bacterial species such as E. coli were significantly decreased (85% versus 64%), as were several Enterococcus spp. (E. faecium and E. casseliflavus) and anaerobes, such as Bacteroides spp. (B. uniformis and B. vulgatus) and Clostridium spp. (C. bifermentans, C. orbiscindens, C. perfringens, and C. symbosium). Conversely, several Bacillus spp. (B. licheniformis, B. mojavensis, and B. pumilus) were significantly more frequent among patients with diarrhea.

Conclusions/Significance

MALDI-TOF is a potentially powerful tool for routine bacterial identification in Africa, allowing for a quick identification of bacterial species.     

Using phenotype microarrays in the assessment of the antibiotic susceptibility profile of bacteria isolated from wastewater in on-site treatment facilities

The scope of the study was to apply Phenotype Biolog MicroArray (PM) technology to test the antibiotic sensitivity of the bacterial strains isolated from on-site wastewater treatment facilities. In the first step of the study, the percentage values of resistant bacteria from total heterotrophic bacteria growing on solid media supplemented with various antibiotics were determined. In the untreated wastewater, the average shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria were 53, 56, and 42%, respectively. Meanwhile, the shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria in the treated wastewater were 39, 33, and 29%, respectively. To evaluate the antibiotic susceptibility of the bacteria present in the wastewater, using the phenotype microarrays (PMs), the most common isolates from the treated wastewater were chosen: Serratia marcescens ss marcescens, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Stenotrophomonas rhizophila, Microbacterium flavescens, Alcaligenes faecalis ss faecalis, Flavobacterium hydatis, Variovorax paradoxus, Acinetobacter johnsonii, and Aeromonas bestiarum. The strains were classified as multi-antibiotic-resistant bacteria. Most of them were resistant to more than 30 antibiotics from various chemical classes. Phenotype microarrays could be successfully used as an additional tool for evaluation of the multi-antibiotic resistance of environmental bacteria and in preliminary determination of the range of inhibition concentration.     

Identification of Selected Putative Oral Pathogens in Primary Root Canal Infections Associated with Symptoms

Overwhelming evidence indicates that bacteria play an essential role in the etiology of different forms of periradicular diseases. The purpose of this study was to assess the prevalence of 11 putative oral pathogens in root canals associated with symptoms using a 16S rDNA-directed polymerase chain reaction (PCR) assay. Associations of the target species in pairs were also recorded. Samples were obtained from the root canals of 20 symptomatic teeth. DNA was extracted from the samples and analysed for the presence of the target bacterial species using PCR. All samples were positive for the presence of bacterial DNA. In general, Treponema denticola was detected in 50% of the cases (ten of 20), Bacteroides forsythus in 40% (eight of 20), Porphyromonas endodontalis in 40% (eight of 20), Porphyromonas gingivalis in 30% (six of 20), Campylobacter rectus in 20% (two of ten), Micromonas micros in 20% (two of ten), Prevotella nigrescens in 10% (two of 20), and Streptococcus anginosus in 10% (one of ten cases). No sample yielded Actinobacillus actinomycetemcomitans, Prevotella intermedia or Fusobacterium nucleatum. The most common bacterial pairs observed between the target species were B. forsythus/P. gingivalis (five cases), B. forsythus/P. endodontalis (four cases), P. endodontalis/P. gingivalis (four cases) andB. forsythus/T. denticola (three cases). The relatively high prevalence of T. denticola, B. forsythus, P. endodontalis, and P. gingivalis suggests that these bacterial species are implicated in the development of symptoms associated with infected root canals.     

Phylogeny of Bacteria Isolated from Rhabditis sp. (Nematoda) and Identification of Novel Entomopathogenic Serratia marcescens Strains

Twenty-five bacterial strains isolated from entomopathogenic nematodes were characterized to the genus level by 16S rRNA phylogeny and BLAST analyses. Bacteria strains isolated could be affiliated with seven genera. Microbacterium-like isolates phylogenetically affiliated with M. oxydans while those of Serratia were highly similar to S. marcescens. 16S rRNA sequences of Bacillus isolates matched those of both B. mycoides and B. weihenstephanesis. One isolate each matched Pseudomonas mosselii, Rheinheimera aquimaris, Achromobacter marplatensis, or Staphylococcus hominis. Serratia isolates were examined further for their pathogenicity to Galleria mellonella larvae. All the Serratia isolates exhibited potent pathogenicity toward G. mellonella larvae and possessed a metalloprotease gene encoding for a novel serralysin-like protein. The nucleotide sequence of the metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions when compared to the closest genBank entry, S. marcescens E-15, with an insertion of a new aspartic acid residue. Tajima’s test for equality of evolutionary rate was significant between the metalloprotease gene sequence of S. marcescens strain DOAB 216-82 (this study) and strain E-15. This new insecticidal metalloprotease gene and/or its product could have applications in agricultural biotechnology.     

Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis,Streptococcus pneumoniae,and Lactococcus lactis for Live Cell Imaging

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.     

Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages

Plant seeds are carriers of both beneficial bacteria and pathogens. Using the 16S rRNA gene clone library technique, we conducted a preliminary study on the community diversity and population succession dynamics of endophytic bacteria in seeds of reciprocal cross hybrid maize at different seed developmental stages. In both hybrid lines (108A and 108B), more types of endophytic bacteria were found at the proembryo-forming stage than in the other two stages, including 29 and 23 bacterial operational taxonomic units (OTUs), respectively. Undibacterium (39.20 and 30.00 % in 108A and 108B, respectively) was the first dominant bacterium to appear. At the milky stage, fewer types of endophytic bacteria in 108A and 108B appeared, including 18 and 16 OTUs, respectively, and the abundance of the dominant genus Burkholderia in the two seed samples reached 73.38 and 80.43 %, respectively. Limnobacter appeared as the second and third endophytic dominant bacterium in 108A (4.55 %) and 108B (5.07 %), respectively, in both seed samples. At the dough stage, the abundance of the first dominant bacterium, Burkholderia, in 108A and 108B was 78.26 and 84.80 %, respectively. Pantoea appeared as the second endophytic dominant bacterium in the both seeds (9.42 and 4.80 % in 108A and 108B, respectively). This is the first study on endophytic bacteria present during several crucial stages of the dynamic grain growth process of plant seeds conducted using culture-independent methods.