Hypothetical Proteins Of Unknown Function Research Articles

Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Multidisciplinary Methods for Screening Toxic Proteins from Phages and Their Potential Molecular Targets.

This chapter presents a comprehensive methodology for the identification, characterization, and functional analyses of potentially toxic hypothetical proteins of unknown function (toxHPUFs) in phages. The methods begin with in vivo toxicity verification of toxHPUFs in bacterial hosts, utilizing conventional drop tests and following growth curves. Computational methods for structural and functional predictions of toxHPUFs are outlined, incorporating the use of tools such as Phyre2, HHpred, and AlphaFold2. To ascertain potential targets, a comparative genomic approach is described using bioinformatics toolkits for sequence alignment and functional annotation. Moreover, steps are provided to predict protein-protein interactions and visualizing these using PyMOL. The culmination of these methods equips researchers with an effective pipeline to identify and analyze toxHPUFs and their potential targets, laying the groundwork for future experimental confirmations.

Discovery of Bactericidal Proteins from Staphylococcus Phage Stab21 Using a High-Throughput Screening Method.

In the escalating battle against antimicrobial resistance, there is an urgent need to discover and investigate new antibiotic strategies. Bacteriophages are untapped reservoirs of such potential antimicrobials. This study focused on Hypothetical Proteins of Unknown Function (HPUFs) from a Staphylococcus phage Stab21. We examined its HPUFs for bactericidal activity against E. coli using a Next Generation Sequencing (NGS)-based approach. Among the 96 HPUFs examined, 5 demonstrated cross-species toxicity towards E. coli, suggesting the presence of shared molecular targets between E. coli and S. aureus. One toxic antibacterial HPUF (toxHPUF) was found to share homology with a homing endonuclease. The implications of these findings are profound, particularly given the potential broad applicability of these bactericidal agents. This study confirms the efficacy of NGS in streamlining the screening process of toxHPUFs, contributes significantly to the ongoing exploration of phage biology, and offers promises in the search for potent antimicrobial agents.

The Oxidative Stress-Induced Hypothetical Protein PG_0686 in Porphyromonas gingivalis W83 Is a Novel Diguanylate Cyclase.

The survival/adaptation of Porphyromonas gingivalis to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Several functional classes of genes, depending on the severity and duration of the exposure, were induced in P. gingivalis under H2O2-induced oxidative stress. The PG_0686 gene was highly upregulated under prolonged oxidative stress. PG_0686, annotated as a hypothetical protein of unknown function, is a 60 kDa protein that carries several domains including hemerythrin, PAS10, and domain of unknown function (DUF)-1858. Although PG_0686 showed some relatedness to several diguanylate cyclases (DGCs), it is missing the classical conserved, active site sequence motif (GGD[/E]EF), commonly observed in other bacteria. PG_0686-related proteins are observed in other anaerobic bacterial species. The isogenic mutant P. gingivalis FLL361 (ΔPG_0686::ermF) showed increased sensitivity to H2O2, and decreased gingipain activity compared to the parental strain. Transcriptome analysis of P. gingivalis FLL361 showed the dysregulation of several gene clusters/operons, known oxidative stress resistance genes, and transcriptional regulators, including PG_2212, CdhR and PG_1181 that were upregulated under normal anaerobic conditions. The intracellular level of c-di-GMP in P. gingivalis FLL361 was significantly decreased compared to the parental strain. The purified recombinant PG_0686 (rPG_0686) protein catalyzed the formation of c-di-GMP from GTP. Collectively, our data suggest a global regulatory property for PG_0686 that may be part of an unconventional second messenger signaling system in P. gingivalis. Moreover, it may coordinately regulate a pathway(s) vital for protection against environmental stress, and is significant in the pathogenicity of P. gingivalis and other anaerobes. IMPORTANCE Porphyromonas gingivalis is an important etiological agent in periodontitis and other systemic diseases. There is still a gap in our understanding of the mechanisms that P. gingivalis uses to survive the inflammatory microenvironment of the periodontal pocket. The hypothetical PG_0686 gene was highly upregulated under prolonged oxidative stress. Although the tertiary structure of PG_0686 showed little relatedness to previously characterized diguanylate cyclases (DGCs), and does not contain the conserved GGD(/E)EF catalytic domain motif sequence, an ability to catalyze the formation of c-di-GMP from GTP is demonstrated. The second messenger pathway for c-di-GMP was previously predicted to be absent in P. gingivalis. PG_0686 paralogs are identified in other anaerobic bacteria. Thus, PG_0686 may represent a novel class of DGCs, which is yet to be characterized. In conclusion, we have shown, for the first time, evidence for the presence of c-di-GMP signaling with environmental stress protective function in P. gingivalis.

Characterization of a Novel Mycovirus from the Phytopathogenic Fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is, globally, one of the most economically important phytopathogenic fungi worldwide, causing the canker and dieback of fruit trees. An increasing number of viruses infecting B. dothidea have lately been reported, several of which could confer hypovirulence. In this study, isolated from strain ZM170285-1 of B. dothidea, a novel double-stranded RNA (dsRNA) mycovirus, tentatively named Botryosphaeria dothidea partitivirus 2 (BdPV2), was identified well. The BdPV2 harbored three dsRNA segments (1–3) with lengths of 1751, 1568, and 1198 bp, which encoded an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), and a hypothetical protein of unknown function, respectively. BLASTp searches revealed that the predicted protein sequences of dsRNA1 and dsRNA2 had the highest identities (74.95% and 61.01%) with the corresponding dsRNAs of Penicillium stoloniferum virus S (PsV-S), whereas dsRNA3 shared the highest identity (32.95%) with the dsRNA3 of Aspergillus ochraceous virus 1 (AoV1). Phylogenetic analysis indicated that BdPV2 belonged to the Gammapartitivirus genus and Partitiviridae family. To our knowledge, this is the first report of a Gammapartitivirus in B. dothidea.

The Transcriptional Regulator MucR, but Not Its Controlled Acid-Activated Chaperone HdeA, Is Essential for Virulence and Modulates Surface Architecture and Properties in Brucella ovis PA.

Brucella ovis is a non-zoonotic bacterium causing contagious epididymitis and other genital lesions in rams and responsible for significant economic losses in sheep-breeding areas. It is a naturally rough (without O-chains in the lipopolysaccharide) Brucella species whose virulence mechanisms have been less explored than those of zoonotic smooth brucellae (bearing O-chains that mask other outer membrane molecules). Considering the rough nature of Brucella ovis, the influence of surface components other than O-chains on its biological properties may be greater than in smooth Brucella species. Here we describe the construction and characterization of the mucR deletion mutant of virulent B. ovis PA, which is defective in a transcriptional regulator, affecting surface properties and virulence in smooth brucellae. This mutant showed increased amounts of three proteins identified as HdeA (acid-activated chaperone), Omp25d (outer membrane protein undetectable in the parental strain), and BOV_A0299 (hypothetical protein of unknown function). This observation correlated with the enhanced transcription of the corresponding genes and constitutes the first report on this type of proteome alteration in Brucella ΔmucR mutants. The upstream regions of the three genes contained AT rich domains with T-A steps described as binding sites for MucR in the Brucella abortus 2308 babR promoter (gene also upregulated in B. ovis ΔmucR), which suggests that hdeA, omp25d, and BOV_A0299 expression could be repressed by MucR through a direct binding to their promoter regions. Relative quantification of transcripts of several other genes selected according to the transcriptome of smooth brucellae ΔmucR mutants revealed not only similarities but also relevant differences among strains, such as those detected in flagellar and virB genes. Periplasmic HdeA has been related to the resistance of B. abortus to acidic pH, conditions encountered by Brucella inside phagocytes, but the deletion of hdeA in B. ovis PA and the ΔmucR mutant did not modify any of the evaluated properties of these strains. The B. ovis PA ΔmucR and ΔmucRΔhdeA mutants had defective in vitro growth and altered surface properties and architecture, exemplified by detectable amounts of Omp25d. Moreover, they showed virulence attenuation but established persistent splenic infection in mice, which encourages their evaluation as specifical attenuated vaccines against B. ovis.

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay.

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.

The BB0345 Hypothetical Protein of Borrelia burgdorferi Is Essential for Mammalian Infection.

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.

Complete genome sequence of a novel mycovirus isolated from the phytopathogenic fungus Corynespora cassiicola in China.

A novel mycovirus, named "Corynespora cassiicola bipartite mycovirus 1" (CcBV1), was isolated from a phytopathogenic fungus, Corynespora cassiicola, the causal agent of rubber leaf fall disease. The nucleotide sequence of the complete genome of CcBV1, which consists of two double-stranded RNA (dsRNA) segments, was determined. The first dsRNA is 2,002bp in length and contains a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) (69kDa), while the second is 1,738bp in length and contains a single ORF encoding a hypothetical protein of unknown function, with an approximately molecular weight of 36kDa. The amino acid sequences of the both deduced proteins are most similar (58.9% and 45.1% identity, respectively) to those of Cryphonectria parasitica bipartite mycovirus 1 (CpBV1). Phylogenetic analysis indicated that CcBV1 clusters together with CpBV1 and other unassigned dsRNA mycoviruses. To the best of our knowledge, this represents the first report of a mycovirus infecting C. cassiicola.

Discovery of Three Toxic Proteins of Klebsiella Phage fHe-Kpn01.

The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.

Crystal structure of a novel fold protein Gp72 from the freshwater cyanophage Mic1.

Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 Å, which represents a novel fold in the α + β class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.

A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins.

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts.

Phenomics reveals a novel putative chloroplast fatty acid transporter in the marine diatom Skeletonema marinoi involved in temperature acclimation

Diatoms are the dominant phytoplankton in temperate oceans and coastal regions and yet little is known about the genetic basis underpinning their global success. Here, we address this challenge by developing the first phenomic approach for a diatom, screening a collection of randomly mutagenized but identifiably tagged transformants. Based upon their tolerance to temperature extremes, several compromised mutants were identified revealing genes either stress related or encoding hypothetical proteins of unknown function. We reveal one of these hypothetical proteins is a novel putative chloroplast fatty acid transporter whose loss affects several fatty acids including the two omega-3, long-chain polyunsaturated fatty acids - eicosapentaenoic and docosahexaenoic acid, both of which have medical importance as dietary supplements and industrial significance in aquaculture and biofuels. This mutant phenotype not only provides new insights into the fatty acid biosynthetic pathways in diatoms but also highlights the future value of phenomics for revealing specific gene functions in these ecologically important phytoplankton.

Complete genome dynamics of a dominant-lineage strain of Xanthomonas oryzae pv. oryzae harbouring a novel plasmid encoding a type IV secretion system.

Xanthomonas oryzae pv. oryzae (Xoo) is a serious pathogen causing bacterial blight disease in rice. Population genomic studies have revealed that rampant inter-strain rather than inter-lineage differences are contributing to the evolutionary success of this pathogen. Here, we report the complete genome sequence of BXO1, a strain of Xoo belonging to a dominant lineage from India. A complete genome-based investigation revealed the presence of two plasmids, pBXO1-1 (66.7 kb) and pBXO1-2 (25.6 kb). The pBXO1-1 plasmid encodes 71 genes, 38 of which encode hypothetical proteins of unknown function. However, these hypothetical genes possess atypical GC content, pointing towards their acquisition and movement through horizontal gene transfer. Interestingly, pBXO1-2 encodes a type IV secretion system (T4SS), which is known to play an important role in the conjugative transfer of genetic material, and also provides fitness to pathogenic bacteria for their enhanced survival. Neither plasmid has been reported previously in any other complete Xoo genome published to date. Our analysis also revealed that the pBXO1-2 plasmid is present in Xanthomonas albilineans str. GPE PC73, which is known to cause leaf scald, a lethal disease in sugarcane. Our complete genome sequence analysis of BXO1 has provided us with detailed insights into the two novel strain-specific plasmids, in addition to decoding their functional capabilities, which were not assessable when using the draft genome sequence of the strain. Overall, our study has revealed the mobility of a novel T4SS in two pathogenic species of Xanthomonas that infect the vascular tissues of two economically important monocot plants, i.e. rice and sugarcane.

Overexpression of YbeD in Escherichia coli Enhances Thermotolerance.

Heat-resistant microbial hosts are required for bioprocess development using high cell density cultivations at the industrial scale. We report that the thermotolerance of Escherichia coli can be enhanced by overexpressing ybeD, which was known to encode a hypothetical protein of unknown function. In the wild-type E. coli BL21(DE3), ybeD transcription level increased over five-fold when temperature was increased from 37°C to either 42°C or 46°C. To study the function of ybeD, a deletion strain and an overexpression strain were constructed. At 46°C, in comparison to the wild type, the ybeD-deletion reduced cell growth half-fold, and the ybeD-overexpression promoted cell growth over two-fold. The growth enhancement by ybeD-overexpression was much more pronounced at 46°C than 37°C. The ybeD-overexpression was also effective in other E. coli strains of MG1655, W3110, DH10B, and BW25113. These findings reveal that ybeD gene plays an important role in enduring high-temperature stress, and that ybeD-overexpression can be a prospective strategy to develop thermotolerant microbial hosts.

Overexpression of YbeD in Escherichia coli Enhances Thermotolerance.

Heat-resistant microbial hosts are required for bioprocess development using high cell density cultivations at the industrial scale. We report that the thermotolerance of Escherichia coli can be enhanced by overexpressing ybeD, which was known to encode a hypothetical protein of unknown function. In the wild type E. coli BL21(DE3), ybeDtranscription level increased over five-fold when temperature was increased from 37°C to either 42°C or 46°C. To study the function of ybeD, a deletion strain and an overexpression strain were constructed. At 46°C, in comparison to the wild type, the ybeD-deletion reduced cell growth half-fold, and the ybeD-overexpression promoted cell growth over two-fold. The growth enhancement by ybeD-overexpression was much more pronounced at 46°C than 37°C. The ybeD-overexpression was also effective in other E. coli strains of MG1655, W3110, DH10B, and BW25113. These findings reveal that ybeD gene plays an important role in enduring high-temperature stress, and that ybeD-overexpression can be a prospective strategy to develop thermotolerant microbial hosts.

Characterization of a novel protein of Leptospira interrogans exhibiting plasminogen, vitronectin and complement binding properties

Leptospirosis is a severe zoonosis caused by pathogenic species of the genus Leptospira. This work focuses on a hypothetical protein of unknown function, encoded by the gene LIC13259, and predicted to be a surface protein, widely distributed among pathogenic leptospiral strain. The gene was amplified from L. interrogans serovar Copenhageni, strain Fiocruz L1-130, cloned and the protein expressed using Escherichia coli as a host system. Immunofluorescence assay showed that the protein is surface-exposed. The recombinant protein LIC13259 (rLIC13259) has the ability to interact with the extracellular matrix (ECM) laminin, in a dose-dependent manner but saturation was not reach. The rLIC13259 protein is a plasminogen (PLG)-binding protein, generating plasmin, in the presence of urokinase PLG-activator uPA. The recombinant protein is able to mediate the binding to human purified terminal complement route vitronectin, C7, C8 and C9, and to recruit and interact with these components from normal human serum (NHS). These interactions are dose-dependent on NHS increased concentration. The binding of rLIC13259 to C8 and vitronectin was slight and pronounced inhibited in the presence of increasing heparin concentration, respectively, suggesting that the interaction with vitronectin occurs via heparin domain. Most interesting, the interaction of rLIC13259 with C9 protein was capable of preventing C9 polymerization, suggesting that the membrane attack complex (MAC) formation was inhibited. Thus, we tentatively assign the coding sequence (CDS) LIC13259, previously annotated as unknown function, as a novel protein that may play an important role in the host’s invasion and immune evasion processes, contributing to the establishment of the leptospiral infection.

Analyzing Phage-Host Protein-Protein Interactions Using Strep-tag® II Purifications.

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host-phage protein-protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host-phage protein-protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

The genome of a novel isolate of Prochlorococcus from the Red Sea contains transcribed genes for compatible solute biosynthesis.

Marine microbes possess genomic and physiological adaptations to cope with varying environmental conditions. So far, the effects of high salinity on the most abundant marine photoautotrophic organism, Prochlorococcus, in marine oligotrophic environments, are mostly unknown. Here, we report the isolation of a new Prochlorococcus strain (RSP50) belonging to high-light (HL) clade II from the Red Sea, one of the warmest and most saline bodies of water in the global oceans. A comparative genomic analysis identified a set of 59 genes that were exclusive to RSP50 relative to currently available Prochlorococcus genomes, the majority of which (70%) encode for hypothetical proteins of unknown function. However, three of the unique genes encode for a complete pathway for the biosynthesis of the compatible solute glucosylglycerol, and are homologous to enzymes found in the sister lineage Synechococcus. Metatranscriptomic analyses of this metabolic pathway in the water column of the Red Sea revealed that the corresponding genes were constitutively transcribed, independent of depth and light, suggesting that osmoregulation using glucosylglycerol is a general feature of HL II Prochlorococcus in the Red Sea.

Generation and In Vivo Characterization of Tn5-Induced Biofilm Mutants of Vibrio cholerae O139.

The Gram-negative bacterium Vibrio cholerae is a unique pathogen with an ability to colonize human intestine as well as outside environments. The biofilm, an organized polymeric structure produced by this bacterium known to be a significant factor for the survival and persistence in hostile conditions. However, the direct role of biofilm formation by this bacterium in environmental persistence, in vivo colonization, and pathogenesis remains unexplored. In this study, we have generated biofilm-altered Tn5 mutants of V. cholerae O139 and evaluated their in vivo colonization ability on mouse model. These Tn5 mutants were found to harbor an independent, single Tn5 insertion in their genome. TheDNA sequence analysis revealed that genomic region wherein Tn5 insertion occurred is identified to be involved in functions like LPS biosynthesis, efflux transporters, motility, purine metabolism, stringent response, VPS synthesis, and a hypothetical protein of unknown function. In single-strain infection with the planktonic culture, the biofilm-altered as well as the biofilm intermediate mutants were found to be more or less similar in their intestinal colonization ability, however infection with their biofilm form, a marked difference was observed between the biofilm deficient and other biofilm forming strains. Further, in the competition experiments, biofilm deficient and proficient mutants were found reduced in their colonization ability and outcompeted by their parent strain. In conclusion, biofilm formation in V. cholerae O139 is a genetically complex process and the controlled and regulated production of biofilm appeared to be necessary for its efficient colonization of mouse intestine.