scholarly journals Chitin Utilization by the Insect-Transmitted Bacterium Xylella fastidiosa

2010 ◽  
Vol 76 (18) ◽  
pp. 6134-6140 ◽  
Author(s):  
Nabil Killiny ◽  
Simone S. Prado ◽  
Rodrigo P. P. Almeida
Keyword(s):  
Carbon Source ◽  
Biofilm Formation ◽  
Xylella Fastidiosa ◽  
Chitinolytic Activity ◽  
Gene Encoding ◽  
Reading Frame ◽  
Biological Assays ◽  
Chitinase A ◽  
Transcriptional Changes

ABSTRACT Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa.

scholarly journals Slipped strand mispairing in the gene encoding sialidase NanH3 in Gardnerella spp

2020 ◽  
Author(s):  
Shakya P. Kurukulasuriya ◽  
Mo H. Patterson ◽  
Janet E. Hill
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Gene Sequence ◽  
Phase Variation ◽  
Cell Wall Proteins ◽  
Vaginal Microbiome ◽  
Gene Encoding ◽  
Reading Frame ◽  
Sialidase Activity ◽  
Mucosal Surfaces

AbstractCell wall proteins with sialidase activity are involved in carbohydrate assimilation, adhesion to mucosal surfaces, and biofilm formation. Gardnerella spp. inhabit the human vaginal microbiome and encode up to three sialidase enzymes, two of which are suspected to be cell wall associated. Here we demonstrate that the gene encoding extracellular sialidase NanH3 is found almost exclusively in G. piotii and closely related Gardnerella genome sp. 3, and its presence correlates with sialidase positive phenotype in a collection of 112 Gardnerella isolates. The nanH3 gene sequence includes a homopolymeric repeat of cytosines that varies in length within cell populations, indicating that this gene is subject to slipped-strand mispairing, a mechanisms of phase variation in bacteria. Variation in the length of the homopolymer sequence results in encoding of either the full length sialidase protein or truncated peptides lacking the sialidase domain due to introduction of reading-frame shifts and premature stop codons. Phase variation in NanH3 may be involved in immune evasion or modulation of adhesion to host epithelial cells, and formation of biofilms characteristic of the vaginal dysbiosis known as bacterial vaginosis.

scholarly journals Deletion of the rpoZ gene, encoding the ω subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1741-1750 ◽  
Author(s):  
Renjith Mathew ◽  
Raju Mukherjee ◽  
Radhakrishnan Balachandar ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Physiological Role ◽  
Wild Type ◽  
Gene Encoding ◽  
Biofilm Growth ◽  
Mutant Bacterium ◽  
Sliding Motility

The ω subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the β′ subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding ω, that the physiological role of the ω subunit also includes providing physical protection to β′. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of ω in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

scholarly journals The Catabolite Repressor/Activator Cra Is a Bridge Connecting Carbon Metabolism and Host Colonization in the Plant Drought Resistance-Promoting Bacterium Pantoea alhagi LTYR-11Z

2018 ◽  
Vol 84 (13) ◽  
Author(s):  
Lei Zhang ◽  
Muhang Li ◽  
Qiqi Li ◽  
Chaoqiong Chen ◽  
Meng Qu ◽  
...  
Keyword(s):  
Carbon Source ◽  
Carbon Metabolism ◽  
Carbon Sources ◽  
Endophytic Bacterium ◽  
Bacterial Attachment ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Host Colonization ◽  
Content Type

ABSTRACT Efficient root colonization is a prerequisite for application of plant growth-promoting (PGP) bacteria in improving health and yield of agricultural crops. We have recently identified an endophytic bacterium, Pantoea alhagi LTYR-11Z, with multiple PGP properties that effectively colonizes the root system of wheat and improves its growth and drought tolerance. To identify novel regulatory genes required for wheat colonization, we screened an LTYR-11Z transposon (Tn) insertion library and found cra to be a colonization-related gene. By using transcriptome (RNA-seq) analysis, we found that transcriptional levels of an eps operon, the ydiV gene encoding an anti-FlhD 4 C 2 factor, and the yedQ gene encoding an enzyme for synthesis of cyclic dimeric GMP (c-di-GMP) were significantly downregulated in the Δ cra mutant. Further studies demonstrated that Cra directly binds to the promoters of the eps operon, ydiV , and yedQ and activates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production and biofilm formation. Consistent with previous findings that Cra plays a role in transcriptional regulation in response to carbon source availability, the activating effects of Cra were much more pronounced when LTYR-11Z was grown within a gluconeogenic environment than when it was grown within a glycolytic environment. We further demonstrate that the ability of LTYR-11Z to colonize wheat roots is modulated by the availability of carbon sources. Altogether, these results uncover a novel strategy utilized by LTYR-11Z to achieve host colonization in response to carbon nutrition in the environment, in which Cra bridges a connection between carbon metabolism and colonization capacity of LTYR-11Z. IMPORTANCE Rapid and appropriate response to environmental signals is crucial for bacteria to adapt to competitive environments and to establish interactions with their hosts. Efficient colonization and persistence within the host are controlled by various regulatory factors that respond to specific environmental cues. The most common is nutrient availability. In this work, we unraveled the pivotal role of Cra in regulation of colonization ability of Pantoea alhagi LTYR-11Z in response to carbon source availability. Moreover, we identified three novel members of the Cra regulon involved in EPS synthesis, regulation of flagellar biosynthesis, and synthesis of c-di-GMP and propose a working model to explain the Cra-mediated regulatory mechanism that links carbon metabolism to host colonization. This study elucidates the regulatory role of Cra in bacterial attachment and colonization of plants, which raises the possibility of extending our studies to other bacteria associated with plant and human health.

scholarly journals Exploitation of a “hockey-puck” phenotype to identify pilus and biofilm regulators in Serratia marcescens through genetic analysis

2016 ◽  
Vol 62 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Robert M.Q. Shanks ◽  
Nicholas A. Stella ◽  
Kimberly M. Brothers ◽  
Denise M. Polaski
Keyword(s):  
Genetic Analysis ◽  
Serratia Marcescens ◽  
Candidate Genes ◽  
Biofilm Formation ◽  
Type I ◽  
Relative Importance ◽  
Gene Encoding ◽  
Uncharacterized Gene ◽  
Mutation Screen

Pili are essential adhesive determinants for many bacterial pathogens. A suppressor mutation screen that takes advantage of a pilus-mediated self-aggregative “hockey-puck” colony phenotype was designed to identify novel regulators of type I pili in Serratia marcescens. Mutations that decreased pilus biosynthesis mapped to the fimABCD operon; to the genes alaT, fkpA, and oxyR; upstream of the flagellar master regulator operon flhDC; and to an uncharacterized gene encoding a predicted DUF1401 domain. Biofilm formation and pilus-dependent agglutination assays were used to characterize the relative importance of the identified genes in pilus biosynthesis. Additional mutagenic or complementation analysis was used to verify the role of candidate genes in pilus biosynthesis. Presented data support a model that CRP negatively regulates pilus biosynthesis through increased expression of flhDC and decreased expression of oxyR. Further studies are warranted to determine the mechanism by which these genes mediate pilus biosynthesis or function.

scholarly journals Biofilm Formation by Psychrobacter arcticus and the Role of a Large Adhesin in Attachment to Surfaces

2013 ◽  
Vol 79 (13) ◽  
pp. 3967-3973 ◽  
Author(s):  
Shannon M. Hinsa-Leasure ◽  
Cassandra Koid ◽  
James M. Tiedje ◽  
Janna N. Schultzhaus
Keyword(s):  
Biofilm Formation ◽  
Time Course ◽  
Microtiter Plate ◽  
Bacterial Attachment ◽  
Wild Type ◽  
Microtiter Plate Assay ◽  
Content Type ◽  
Reading Frame ◽  
Transposon Mutants

ABSTRACTPsychrobacter arcticusstrain 273-4, an isolate from a Siberian permafrost core, is capable of forming biofilms when grown in minimal medium under laboratory conditions. Biofilms form at 4 to 22°C when acetate is supplied as the lone carbon source and with 1 to 7% sea salt.P. arcticusis also capable of colonizing quartz sand. Transposon mutagenesis identified a gene important for biofilm formation byP. arcticus. Four transposon mutants were mapped to a 20.1-kbp gene, which is predicted to encode a protein of 6,715 amino acids (Psyc_1601). We refer to this open reading frame ascat1, for cold attachment gene 1. Thecat1mutants are unable to form biofilms at levels equivalent to that of the wild type, and there is no impact on the planktonic growth characteristics of the strains, indicating a specific role in biofilm formation. Through time course studies of the static microtiter plate assay, we determined thatcat1mutants are unable to form biofilms equivalent to that of the wild type under all conditions tested. In flow cell experiments,cat1mutants initially are unable to attach to the surface. Over time, however, they form microcolonies, an architecture very different from that produced by wild-type biofilms. Our results demonstrate that Cat1 is involved in the initial stages of bacterial attachment to surfaces.

scholarly journals Molecular and Functional Characterization of theRhodopseudomonas palustris No. 7 Phosphoenolpyruvate Carboxykinase Gene

1999 ◽  
Vol 181 (9) ◽  
pp. 2689-2696 ◽  
Author(s):  
Masayuki Inui ◽  
Kaori Nakata ◽  
Jung Hyeob Roh ◽  
Kenneth Zahn ◽  
Hideaki Yukawa
Keyword(s):  
Carbon Source ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Transcriptional Start Site ◽  
Rhodopseudomonas Palustris ◽  
Functional Characterization ◽  
Carbon Sources ◽  
Pcr Amplification ◽  
Upstream Sequence ◽  
Gene Encoding ◽  
Reading Frame

ABSTRACT The pckA gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacteriumRhodopseudomonas palustris No. 7. Sequencing of a 2.5-kb chromosomal SmaI-PstI fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known pckA genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of thepckA initiation codon and an upstream sequence similar to ς70 promoters. Studies of a pckA-lacZ gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, thepckA gene was induced in log phase, irrespective of the carbon source. A R. palustris No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C4-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in R. palustris No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.

scholarly journals Slipped strand mispairing in the gene encoding sialidase NanH3 in Gardnerella spp.

2020 ◽  
Author(s):  
Shakya P. Kurukulasuriya ◽  
Mo H. Patterson ◽  
Janet E. Hill
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Gene Sequence ◽  
Phase Variation ◽  
Cell Wall Proteins ◽  
Vaginal Microbiome ◽  
Gene Encoding ◽  
Reading Frame ◽  
Sialidase Activity ◽  
Mucosal Surfaces

Cell wall proteins with sialidase activity are involved in carbohydrate assimilation, adhesion to mucosal surfaces, and biofilm formation. Gardnerella spp. inhabit the human vaginal microbiome and encode up to three sialidase enzymes, two of which are suspected to be cell wall associated. Here we demonstrate that the gene encoding extracellular sialidase NanH3 is found almost exclusively in G. piotii and closely related Gardnerella genome sp. 3, and its presence correlates with sialidase positive phenotype in a collection of 112 Gardnerella isolates. The nanH3 gene sequence includes a homopolymeric repeat of cytosines that varies in length within cell populations, indicating that this gene is subject to slipped-strand mispairing, a mechanisms of phase variation in bacteria. Variation in the length of the homopolymer sequence results in encoding of either the full length sialidase protein or truncated peptides lacking the sialidase domain due to introduction of reading-frame shifts and premature stop codons. Phase variation in NanH3 may be involved in immune evasion or modulation of adhesion to host epithelial cells, and formation of biofilms characteristic of the vaginal dysbiosis known as bacterial vaginosis.

scholarly journals Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

1999 ◽  
Vol 181 (3) ◽  
pp. 849-857 ◽  
Author(s):  
P. Aneja ◽  
T. C. Charles
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Start Codon ◽  
Start Site ◽  
Gene Encoding ◽  
Reading Frame ◽  
Isolation And Characterization

ABSTRACT We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.

scholarly journals 2604. Changes in a Fatty Acid Kinase Associated with Daptomycin (DAP) Resistance Lead to Increased Collagen Binding and Biofilm Formation in Enterococcus faecalis

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S905-S905
Author(s):  
William R Miller ◽  
Kavindra V Singh ◽  
Jinnethe Reyes ◽  
Barbara E Murray ◽  
Cesar A Arias
Keyword(s):  
Extracellular Matrix ◽  
Fatty Acid ◽  
Biofilm Formation ◽  
Type I Collagen ◽  
Treatment Options ◽  
Type I ◽  
Gene Encoding ◽  
Stress Response System ◽  
Healthcare Associated

Abstract Background Enterococci are a major cause of healthcare-associated infections with limited treatment options. We previously identified that mutations in dak (a gene encoding a putative fatty acid kinase), ace (a collagen adhesin) and the YxdJK stress response system are associated with DAP resistance (DAP-R) in E. faecalis (Efs) in the absence of a functional LiaFSR system. Here, we examined the role of DAK in pathogenesis by examining the ability of the mutants to produce biofilm and bind to collagen, an important protein of the extracellular matrix. Methods Previously, the Efs strain OG1RFΔliaR (inactive LiaFSR system, DAP susceptible) was adapted to make a DAP-R derivative (mutations in yxdK, dak, and ace), and the mutant OG1RFΔliaRΔc-dak, lacking the C-terminal domain of dak, and its complement OG1RFΔliaRΔc-dak::c-dak were constructed to study the dak mutation in isolation. Biofilm formation (BF) for the above strains was assayed after growth in tryptic soy broth with glucose in 96-well plates at 37° C for 24 hours. Bacteria were fixed with Bouin’s fixative, stained with crystal violet, and biofilm was quantitated by absorbance at 570 nm. For collagen adherence, 96-well plates were coated with 10 μg/well type I collagen, with 2% bovine serum albumin (BSA) as a control. Bacteria grown at 46° C (to induce ace expression) were added at OD 600 nm of 1.0 and allowed to bind for 2 hours. Non-adherent bacteria were removed by washing, cells were fixed, stained, and quantified as above. Results When compared with OG1RFΔliaR, the DAP-R derivative exhibited a significant increase in BF (4.6 vs. 2.4, P < 0.001). This enhanced biofilm phenotype was also seen in the OG1RFΔliaRΔc-dak mutant (6.1 vs. 2.4, P < 0.001), and reverted on complementation of the full-length dak in its native chromosomal location (2.4 vs. 2.6, p = 0.72). DAK was also found to impact adherence to collagen, with OG1RFΔliaRΔc-dak showing increased binding to collagen when compared with OG1RFΔliaR (7.9 vs. 3.4, P < 0.001), a phenotype which reverted on complementation (7.9 vs. 1.2, P < 0.001). Conclusion Changes in an enzyme involved in DAP adaptation lead to biofilm formation and adherence to extracellular matrix proteins, potentially enhancing virulence in the setting of DAP-R. Disclosures All authors: No reported disclosures.

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