Genomic Insights of an Andean Multi-resistant Soil Actinobacterium of Biotechnological Interest

ABSTRACTCentral Andean Ecosystems (between 2000 and 6000 masl) are typical arid to semiarid environments suffering from the highest total solar and UVB radiation on the planet but displaying numerous salt flats and shallow lakes. Isolated from these environments, Andean Microbial Communities (AME) of exceptional biodiversity endures multiple severe conditions. Also, the poly-extremophilic nature of AME’s microbes indicates the potential for biotechnological applications. In this context, the presented study used genome mining and physiological characterization to reveal the multi-resistant profile of Nesterenkonia sp. Act20, an actinobacterium isolated from the soil surrounding Lake Socompa, Salta, Argentina (3570 m). UV-B, desiccation, and copper assays showed the strain’s exceptional resistance to all these factors. Act20’s genome presented coding sequences involving antibiotics, low temperatures, UV and arsenic resistance, nutrient limiting conditions, osmotic stress response, low atmospheric oxygen pressure, heavy metal stress, and resistance to fluoride and chlorite. Act20 can also synthesize proteins and natural products such as an insecticide, bacterial cellulose, ectoine, bacterial hemoglobin, and even antibiotics like colicin V and aurachin C. We also found numerous enzymes for animal and vegetal biomass degradation and application in other industrial processes.The herein report shed light on the microbial adaptation to high-altitude environments, its possible extrapolation for studying other extreme environments of relevance, and its application to industrial and biotechnological processes.HIGHLIGHTSArid Andean Soils are attractive sources of microbial strains useful in biotechnological processes.Physiological studies revealed the multi-resistant nature of the poly-extremophile Nesterenkonia sp. Act20.Act20’s genome analysis showed a complete set of genes coding for proteins involved in resistance to multiple stresses, including extremoenzymes and extremolytes.

Novel Probiotic Mechanisms of the Oral Bacterium Streptococcus sp. A12 as Explored with Functional Genomics

ABSTRACT Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans. A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual and was shown to interfere potently with the growth and virulence properties of S. mutans. In this study, we applied functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans. Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to that of the parental strain. The A12 pcfO mutant lost the ability to inhibit comX-inducing peptide (XIP) signaling by S. mutans, while mutants with changes in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans. Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiological factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health. IMPORTANCE Advances in defining the composition of health-associated biofilms have highlighted the important role of beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological bases underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans using various gene products with diverse functions. A12 displayed enhanced competitiveness by (i) disrupting intercellular communication pathways of S. mutans, (ii) sensing and resisting antimicrobial peptides, and (iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

Exploring novel probiotic mechanisms ofStreptococcusA12 with functional genomics

Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, likeStreptococcus mutans.A novel oral bacterium, designatedStreptococcusA12, was previously isolated from supragingival dental plaque of a caries-free individual, and was shown to interfere potently with the growth and virulence properties ofS. mutans. Here, we apply functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of,S. mutans.Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete withS. mutanswere identified. Selected genes, designated aspotentialcompetitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete withS. mutanscompared to the parental strain. The A12pcfOmutant lost the ability to inhibitcomX-inducingpeptide (XIP) signaling byS. mutans, while mutants in thepcfFEGlocus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism ofS. mutans.Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiologic factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.ImportanceAdvances in defining the composition of health-associated biofilms have highlighted the important role for beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological basis underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate,StreptococcusA12, to compete with the dental pathogenStreptococcus mutans, using various gene products with diverse functions. A12 displayed enhanced competitiveness by: i) disrupting intercellular communication pathways ofS. mutans, ii) sensing and resisting antimicrobial peptides, and iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed byStreptococcusA12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

Insights into the Genome Sequence ofChromobacterium amazonenseIsolated from a Tropical Freshwater Lake

Members of the genusChromobacteriumhave been isolated from geographically diverse ecosystems and exhibit considerable metabolic flexibility, as well as biotechnological and pathogenic properties in some species. This study reports the draft assembly and detailed sequence analysis ofChromobacterium amazonensestrain 56AF. The de novo-assembled genome is 4,556,707 bp in size and contains 4294 protein-coding and 95 RNA genes, including 88 tRNA, six rRNA, and one tmRNA operon. A repertoire of genes implicated in virulence, for example, hemolysin, hemolytic enterotoxins, colicin V, lytic proteins, and Nudix hydrolases, is present. The genome also contains a collection of genes of biotechnological interest, including esterases, lipase, auxins, chitinases, phytoene synthase and phytoene desaturase, polyhydroxyalkanoates, violacein, plastocyanin/azurin, and detoxifying compounds. Importantly, unlike otherChromobacteriumspecies, the 56AF genome contains genes for pore-forming toxin alpha-hemolysin, a type IV secretion system, among others. The analysis of theC. amazonensestrain 56AF genome reveals the versatility, adaptability, and biotechnological potential of this bacterium. This study provides molecular information that may pave the way for further comparative genomics and functional studies involvingChromobacterium-related isolates and improves our understanding of the global genomic diversity ofChromobacteriumspecies.

Draft Genome Sequence of the Oral Commensal Streptococcus oralis 89a with Interference Activity against Respiratory Pathogens

We report the draft genome sequence of the oral commensal Streptococcus oralis 89a isolated from the throat of a healthy child during a streptococcal tonsillitis outbreak in Umeå, Sweden. S. oralis 89a was known to have interference activity against respiratory pathogens in which the colicin V was the potential bacteriocin-encoding gene.

Virulence Profiles, Phylogenetic Background, and Antibiotic Resistance ofEscherichia coliIsolated from Turkeys with Airsacculitis

Avian PathogenicEscherichia coli(APEC) has been studied for decades because of its economic impact on the poultry industry. Recently, the zoonotic potential of APEC and multidrug-resistant strains have emerged. The aim of this study was to characterize 225 APEC isolated from turkeys presenting airsacculitis. The results showed that 92% of strains presented a multidrug-resistance (MDR), and the highest levels of resistance were to sulfamethazine (94%) and tetracycline (83%). Half of these strains were classified in phylogenetic group B2, followed by B1 (28.6%), A (17.1%), and D (4.8%). The prevalence of virulence genes was as follows: salmochelin (iroN,95%), increased serum survival (iss,93%), colicin V (cvi/cva,67%), aerobactin (iucD,67%), temperature-sensitive haemagglutinin (tsh,56%), iron-repressible protein (irp2,51%), invasion brain endothelium (ibeA,31%), vacuolating autotransporter toxin (vat,24%), K1 antigen (neuS,19%), enteroaggregative heat-stable cytotoxin (astA,17%), and pilus associated with pyelonephritis (papC,15%). These results demonstrate that the majority of the investigated strains belonged to group B2 and were MDR. These data suggest that turkeys may serve as a reservoir of pathogenic and multidrug-resistance strains, reinforcing the idea that poultry plays a role in the epidemiological chain of ExPEC.

An Escherichia coli Nissle 1917 Missense Mutant Colonizes the Streptomycin-Treated Mouse Intestine Better than the Wild Type but Is Not a Better Probiotic

ABSTRACTPreviously we reported that the streptomycin-treated mouse intestine selected for two differentEscherichia coliMG1655 mutants with improved colonizing ability: nonmotileE. coliMG1655flhDCdeletion mutants that grew 15% fasterin vitroin mouse cecal mucus and motileE. coliMG1655envZmissense mutants that grew slowerin vitroin mouse cecal mucus yet were able to cocolonize with the faster-growingflhDCmutants. TheE. coliMG1655envZgene encodes a histidine kinase that is a member of theenvZ-ompRtwo-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, theenvZP41Lgene was transferred from the intestinally selectedE. coliMG1655 mutant toE. coliNissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both theE. coliMG1655 andE. coliNissle 1917 strains containingenvZP41Lproduced more phosphorylated OmpR than their parents. TheE. coliNissle 1917 strain containingenvZP41Lalso became more resistant to bile salts and colicin V and grew 50% slowerin vitroin mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer thanE. coliNissle 1917. However,E. coliNissle 1917envZP41Lwas not better at preventing colonization by enterohemorrhagicE. coliEDL933. The data can be explained according to our “restaurant” hypothesis for commensalE. colistrains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invadingE. colipathogens planktonically.

RpfF-Dependent Regulon of Xylella fastidiosa

Xylella fastidiosa regulates traits important to both virulence of grape as well as colonization of sharpshooter vectors via its production of a fatty acid signal molecule known as DSF whose production is dependent on rpfF. Although X. fastidiosa rpfF mutants exhibit increased virulence to plants, they are unable to be spread from plant to plant by insect vectors. To gain more insight into the traits that contribute to these processes, a whole-genome Agilent DNA microarray for this species was developed and used to determine the RpfF-dependent regulon by transcriptional profiling. In total, 446 protein coding genes whose expression was significantly different between the wild type and an rpfF mutant (false discovery rate < 0.05) were identified when cells were grown in PW liquid medium. Among them, 165 genes were downregulated in the rpfF mutant compared with the wild-type strain whereas 281 genes were over-expressed. RpfF function was required for regulation of 11 regulatory and σ factors, including rpfE, yybA, PD1177, glnB, rpfG, PD0954, PD0199, PD2050, colR, rpoH, and rpoD. In general, RpfF is required for regulation of genes involved in attachment and biofilm formation, enhancing expression of hemagglutinin genes hxfA and hxfB, and suppressing most type IV pili and gum genes. A large number of other RpfF-dependent genes that might contribute to virulence or insect colonization were also identified such as those encoding hemolysin and colicin V, as well as genes with unknown functions.