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.     

Microbial interactions in the mosquito gut determine Serratia colonization and blood-feeding propensity

How microbe–microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host–microbe interactions and provides evidence that microbial interactions influence mosquito behavior.Subject terms: Symbiosis, Microbial ecology, Bacterial host response     

Pyrazines Responsible for the Potatolike Odor Produced by Some Serratia and Cedecea Strains

The pyrazines responsible for the potatolike odor produced by some Serratia and Cedecea cultures were identified by gas-liquid chromatography (with flame ionization and thermoionic ionization detectors) and mass spectrometry. Alkylpyrazines were produced by the six strains studied irrespective of their odors. The major alkyl-methoxypyrazine produced by Cedecea davisae was 3-sec-butyl-2-methoxypyrazine, and that produced by odoriferous Serratia strains (S. rubidaea, S. odorifera, and S. ficaria) was 3-isopropyl-2-methoxy-5-methyl-pyrazine. Other pyrazines produced by some strains were 3-isopropyl-2-methoxypyrazine, 3-isobutyl-2-methoxypyrazine, 3-sec-butyl-2-methoxy-5-methylpyrazine, and 3-isobutyl-2-methoxy-6-methylpyrazine. Some of these pyrazines had not previously been found as natural products or to be produced by bacteria.     

Role of bacterial pyrroloquinoline quinone in phosphate solubilizing ability and in plant growth promotion on strain <Emphasis Type="Italic">Serratia</Emphasis> sp. S119

The aim of this study was to analyze if cofactor pyrroquinoline quinone from Serratia sp. S119 is involved in the inorganic phosphate solubilization mechanism and in its ability to promote the plant growth. Site directed mutagenesis was performed to obtain a pqqE- minus mutant of strain Serratia sp. S119. The phosphate solubilization ability, gluconate and PQQ production of the mutant Serratia sp. RSL (pqqE-) was analyzed. Mutant RSL (pqqE-) showed significant decrease in P soluble and gluconic acid levels produced and undetectable levels of PQQ cofactor compared with wild-type strain. Complementation with synthetic PQQ cofactor restored P solubilization and gluconate production reaching the levels produced by wild-type strain. PqqE gene sequence indicated that it is highly conserved within Serratia strains and its product shows conserved motifs found in other PqqE proteins of several bacteria. The effect of the inoculation of the PQQ- mutant on peanut and maize plants was evaluated in pot assays. Plants growth parameters showed no differences among the different treatments indicating that PQQ from Serratia sp. S119 is not involved in the growth promotion of these plants. PQQ cofactor is essential for phosphate solubilization ability of Serratia sp. S119 but is not required for growth promotion of peanut and maize plants.     

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae

Background

Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity.

Results

We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99 % sequence identity in rDNA sequence and orthology across 85.6 % of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8 %) were present in Serratia while 33 (84.6 %) and 35 (89 %) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively.

Conclusion

The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result – killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1697-8) contains supplementary material, which is available to authorized users.     

Comparative Genomics of Serratia spp.: Two Paths towards Endosymbiotic Life

Symbiosis is a widespread phenomenon in nature, in which insects show a great number of these associations. Buchnera aphidicola, the obligate endosymbiont of aphids, coexists in some species with another intracellular bacterium, Serratia symbiotica. Of particular interest is the case of the cedar aphid Cinara cedri, where B. aphidicola BCc and S. symbiotica SCc need each other to fulfil their symbiotic role with the insect. Moreover, various features seem to indicate that S. symbiotica SCc is closer to an obligate endosymbiont than to other facultative S. symbiotica, such as the one described for the aphid Acirthosyphon pisum (S. symbiotica SAp). This work is based on the comparative genomics of five strains of Serratia, three free-living and two endosymbiotic ones (one facultative and one obligate) which should allow us to dissect the genome reduction taking place in the adaptive process to an intracellular life-style. Using a pan-genome approach, we have identified shared and strain-specific genes from both endosymbiotic strains and gained insight into the different genetic reduction both S. symbiotica have undergone. We have identified both retained and reduced functional categories in S. symbiotica compared to the Free-Living Serratia (FLS) that seem to be related with its endosymbiotic role in their specific host-symbiont systems. By means of a phylogenomic reconstruction we have solved the position of both endosymbionts with confidence, established the probable insect-pathogen origin of the symbiotic clade as well as the high amino-acid substitution rate in S. symbiotica SCc. Finally, we were able to quantify the minimal number of rearrangements suffered in the endosymbiotic lineages and reconstruct a minimal rearrangement phylogeny. All these findings provide important evidence for the existence of at least two distinctive S. symbiotica lineages that are characterized by different rearrangements, gene content, genome size and branch lengths.     

Evolution of virulence in a novel family of transmissible mega-plasmids

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; a mber d isease-a ssociated p lasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.     

Enteric bacteria of field-collected Colorado potato beetle larvae inhibit growth of the entomopathogens Photorhabdus temperata and Beauveria bassiana

The nematode Heterorhabditis marelatus fails to reproduce in the Colorado potato beetle, Leptinotarsa decemlineata, possibly due to interference from the enteric bacteria of the beetle. Specifically, the enteric bacteria inhibit the growth of Photorhabdus temperata, the enteric symbiont of the nematode, in vitro. However, previous work was based on a laboratory culture of L. decemlineata, and we wished to determine if similar bacteria were present in the field. Therefore, we cultured the enteric bacteria of fourth-instar larvae collected from the field at two locations in Maryland and Virginia. Representatives of the genera Pantoea, Enterobacter, Pseudomonas, Acinetobacter, Serratia, Stenotrophomonas, Curtobacterium, Bacillus, Lactococcus and Enterococcus were identified by sequencing of their 16S rDNA. Isolates belonging to the genera Pantoea, Enterobacter, Pseudomonas, Serratia and Bacillus inhibited the growth of P. temperata. A number of these isolates also inhibited the entomopathogenic fungus Beauveria bassiana in vitro.     

Food color ‘Azorubine’ interferes with quorum sensing regulated functions and obliterates biofilm formed by food associated bacteria: An in vitro and in silico approach

Quorum sensing (QS) plays a crucial role in different stages of biofilm development, virulence production, and subsequently to the growth of bacteria in food environments. Biofilm mediated spoilage of food is one of the ongoing challenge faced by the food industry worldwide as it incurs substantial economic losses and leads to various health issues. In the present investigation, we studied the interference of quorum sensing, its regulated virulence functions, and biofilm in food-associated bacteria by colorant azorubine. In vitro bioassays demonstrated significant inhibition of QS and its coordinated virulence functions in Chromobacterium violaceum 12472 (violacein) and Pseudomonas aeruginosa PAO1 (elastase, protease, pyocyanin, and alginate). Further, the decrease in the production EPS (49–63%) and swarming motility (61–83%) of the pathogens was also recorded at sub-MICs. Azorubine demonstrated broad-spectrum biofilm inhibitory potency (50–65%) against Chromobacterium violaceum, Pseudomonas aeruginosa, E. coli O157:H7, Serratia marcescens, and Listeria monocytogenes. ROS generation due to the interaction between bacteria and azorubine could be responsible for the biofilm inhibitory action of the food colorant. Findings of the in vitro studies were well supported by molecular docking and simulation analysis of azorubine and QS virulence proteins. Azorubine showed strong binding to PqsA as compared to other virulent proteins (LasR, Vfr, and QscR). Thus, it is concluded that azorubine is a promising candidate to ensure food safety by curbing the menace of bacterial QS and biofilm-based spoilage of food and reduce economic losses.     

Enzymes of Tryptophan Biosynthesis in Serratia marcescens

In Serratia marcescens, the tryptophan biosynthetic enzymes were formed coordinately. A number of tryptophan auxotrophs showed single biochemical lesions; several mutants showed pleiotropic effects. Sucrose density gradient centrifugation revealed an unique pattern of migration of the tryptophan biosynthetic enzymes. The repression response of the Serratia enzymes to exogenous tryptophan was fivefold more sensitive than that found in Escherichia coli. When this information is contrasted with the available information on the other Enterobacteriaceae, one is compelled to conclude that S. marcescens enjoys a rather marked evolutionary divergence from the other enteric organisms.     

Bacterial Community Diversity in the Brazilian Atlantic Forest Soils

The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29–54%), Proteobacteria (16–38%), and Verrucomicrobia (0.6–14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41–152) and species (263–446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria.     

Survey of Plant Drought-Resistance Promoting Bacteria from Populus euphratica Tree Living in Arid Area

Two hundred and thirty-two bacterial strains were isolated from the rhizospheric soil of Populus euphratica which is the dominant tree living in extreme arid regions in northwest China. Some strains with plant growth-promoting bacteria related metabolic characteristics were able to promote drought resistance in plants after inoculation. Ten strains with the greatest effects increased the dry weight of wheat shoots from 0.5 to 34.4 %, and the surface area of the root systems from 12.56 to 212.17 % compared to the control after drought treatment whereas no obvious promoting effect was observed in normal water conditions. These 10 strains were identified to be of the genera Pseudomonas, Bacillus, Stenotrophomonas and Serratia by 16S rRNA (rrs) gene sequence alignment. Among these strains, Serratia sp. 1-9 and Pseudomonas sp. 5-23 were the two most effective strains. Both of them produced auxin and the production increased significantly when cultured under simulated drought conditions which are inferred to be the most plausible mechanism for their plant growth-promoting effect under drought stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0479-3) contains supplementary material, which is available to authorized users.     

Symbiotic microbial studies in diverse populations of Aphis gossypii,existing on altered host plants in different localities during different times

Complex interactions between symbiotic bacteria and insects ultimately result in equilibrium in all aspects of life in natural insect populations. In this study, abundance of principal symbiotic bacteria was estimated using qPCR in 1553 individuals of aphids, Aphis gossypii. Aphids were sampled from primary and secondary host plants—hibiscus and cotton. Hibiscus aphids were collected from 24 different locations in April, September, and November, whereas cotton aphids were collected between 2015 and 2017 from areas with wide variations in climatic conditions. About 30%–45% aphids were recorded with the most dominant symbiont, Arsenophonus. The other symbionts were in low frequency, and about 7% of aphids were noted with Hamiltonella, Acinetobacter, and Microbacterium, and 3% of aphids were verified with Serratia and Pseudomonas. Aphids infected with Hamiltonella, Arsenophonus, and Serratia can influence Buchnera densities. Hamiltonella has positive interaction with densities of Arsenophonus and Serratia. Almost 100% coinfection of Hamiltonella and Arsenophonus was detected in Xinxiang aphids and 50% coinfection was reported in aphids from North China, while no coinfection was detected in Hainan aphids. These findings describe the prevalence pattern and richness of core community of symbiotic bacteria in naturally occurring populations of A. gossypii and provide new insights for the study of symbiotic bacteria.     

Identification of Genes Contributing to the Virulence of Francisella tularensis SCHU S4 in a Mouse Intradermal Infection Model

Background

Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis.

Methodology/Principal Findings

The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD₅₀ of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably ΔpyrB and ΔrecA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD₅₀ of >10³ CFU. In fact, the latter seven mutants showed very marked attenuation with an LD₅₀ of ≥10⁷ CFU.

Conclusions/Significance

The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis.     

Enterobacteriaceae and Pseudomonas aeruginosa Recovered from Vegetable Salads

Klebsiella, Enterobacter, and Serratia were recovered frequently in high counts from vegetable salads. Pseudomonas aeruginosa, although isolated frequently, was in lower counts.     

Inhibition of Quorum Sensing Mediated Virulence Factors Production in Urinary Pathogen Serratia marcescens PS1 by Marine Sponges

The focal intent of this study was to find out an alternative strategy for the antibiotic usage against bacterial infections. The quorum sensing inhibitory (QSI) activity of marine sponges collected from Palk Bay, India was evaluated against acyl homoserine lactone (AHL) mediated violacein production in Chromobacterium violaceum (ATCC 12472), CV026 and virulence gene expressions in clinical isolate Serratia marcescens PS1. Out of 29 marine sponges tested, the methanol extracts of Aphrocallistes bocagei (TS 8), Haliclona (Gellius) megastoma (TS 25) and Clathria atrasanguinea (TS 27) inhibited the AHL mediated violacein production in C. violaceum (ATCC 12472) and CV026. Further, these sponge extracts inhibited the AHL dependent prodigiosin pigment, virulence enzymes such as protease, hemolysin production and biofilm formation in S. marcescens PS1. However, these sponge extracts were not inhibitory to bacterial growth, which reveals the fact that the QSI activity of these extracts was not related to static or killing effects on bacteria. Based on the obtained results, it is envisaged that the marine sponges could pave the way to prevent quorum sensing (QS) mediated bacterial infections.