R. Dwayne Lunsford

The Human Microbiome Project (HMP), funded as an initiative of the NIH Roadmap for Biomedical Research ( http://nihroadmap.nih.gov ), is a multi-component community resource. The goals of the HMP are: (1) to take advantage of new, high-throughput technologies to characterize the human microbiome more fully by studying samples from multiple body sites from each of at least 250 “normal” volunteers; (2) to determine whether there are associations between changes in the microbiome and health/disease by studying several different medical conditions; and (3) to provide both a standardized data resource and new technological approaches to enable such studies to be undertaken broadly in the scientific community. The ethical, legal, and social implications of such research are being systematically studied as well. The ultimate objective of the HMP is to demonstrate that there are opportunities to improve human health through monitoring or manipulation of the human microbiome. The history and implementation of this new program are described here.

Serratia marcescens is an environmental gram-negative bacterium that causes invasive disease in rare cases. During 2020-2022, an outbreak of 21 invasive Serratia infections occurred in a prison in California, USA. Most (95%) patients had a history of recent injection drug use (IDU). We performed whole-genome sequencing and found isolates from 8 patients and 2 pieces of IDU equipment were closely related. We also identified social interactions among patients. We recovered S. marcescens from multiple environmental samples throughout the prison, including personal containers storing Cell Block 64 (CB64), a quaternary ammonium disinfectant solution. CB64 preparation and storage conditions were suboptimal for S. marcescens disinfection. The outbreak was likely caused by contaminated CB64 and propagated by shared IDU equipment and social connections. Ensuring appropriate preparation, storage, and availability of disinfectants and enacting interventions to counteract disease spread through IDU can reduce risks for invasive Serratia infections in California prisons.

Systematic analysis of the entire two-component signal transduction system (TCSTS) gene complement of Staphylococcus aureus revealed the presence of a putative TCSTS (designated SrhSR) which shares considerable homology with the ResDE His-Asp phospho-relay pair of Bacillus subtilis. Disruption of the srhSR gene pair resulted in a dramatic reduction in growth of the srhSR mutant, when cultured under anaerobic conditions, and a 3-log attenuation in growth when analyzed in the murine pyelonephritis model. To further understand the role of SrhSR, differential display two-dimensional gel electrophoresis was used to analyze the cell-free extracts derived from the srhSR mutant and the corresponding wild type. Proteins shown to be differentially regulated were identified by mass spectrometry in combination with protein database searching. An srhSR deletion led to changes in the expression of proteins involved in energy metabolism and other metabolic processes including arginine catabolism, xanthine catabolism, and cell morphology. The impaired growth of the mutant under anaerobic conditions and the dramatic changes in proteins involved in energy metabolism shed light on the mechanisms used by S. aureus to grow anaerobically and indicate that the staphylococcal SrhSR system plays an important role in the regulation of energy transduction in response to changes in oxygen availability. The combination of proteomics, bio-informatics, and microbial genetics employed here represents a powerful set of techniques which can be applied to the study of bacterial gene function.

Attempted allelic replacement of 144 <i>Streptococcus pneumoniae</i> open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes <i>(obg</i>, <i>spoIIIJ2</i>, <i>trmU</i>, <i>yacA</i>, <i>yacM</i>, <i>ydiC</i>, <i>ydiE</i>, <i>yjbN</i>, <i>yneS</i>, <i>yphC</i>, <i>ysxC</i>, <i>ytaG</i>, <i>yloI</i> and <i>yxeH4)</i> were also essential in <i>Staphylococcus aureus</i> and <i>Haemophilus influenzae </i>or<i> Escherichia coli,</i> 2 genes <i>(yrrK</i> and <i>ydiB)</i> were only essential in <i>H. influenzae</i> as well as <i>S. pneumoniae</i> and 8 genes were necessary for growth of <i>S.</i><i>pneumoniae</i> and <i>S. aureus </i>and did not have a homolog in <i>H. influenzae</i><i>(murD2</i>, <i>ykqC</i>, <i>ylqF</i>, <i>yqeH</i>, <i>ytgP</i>, <i>yybQ)</i> or were not essential in that organism <i>(yqeL, yhcT)</i>. The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including <i>Bacillus subtilis</i>, <i>E. coli</i> and <i>Mycoplasma </i>sp<i>.</i> demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.

Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.

ABSTRACT Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus . In this study we showed through genetic disruption experiments that mvaA , which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus . Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli . The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K i of 320 μM, over 10 4 higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

In the last two decades, the search for completely novel antibacterial agents has acquired a new sense of urgency due to the remarkable rise of antibiotic resistance among key bacterial pathogens. More recently, the advent of bacterial genomics has provided investigators with the data and bioinformatic tools to rationally identify novel antibacterial targets and the genome-scaled methodologies to validate them. Only 6 years have elapsed since the publication of the first complete bacterial genome sequence, but more than 50 complete microbial genome sequences are now available. This review will discuss the advantages and limitations of the existing bacterial genome dataset for the rational identification of novel antibacterial targets. Since the ability to rapidly identify essential genes where loss of function is coincident with loss of viability is the most important task of genomics-based target validation, essentiality testing methodologies (in which molecular genetic techniques are used to determine whether or not a gene product is required for viability of the parent cell) will be surveyed and their amenability to genome-scaled analysis assessed. Finally, we will discuss the impact of bacterial genomics to date on the development of novel and effective antibiotics.

ABSTRACT Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn 916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei ). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded d -alanine- d -alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of d -alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137–4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that d -alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.

Streptococcus gordonii Wicky becomes competent only after stimulation with conditioned medium from strain Challis as a source of competence factor (CF). A 3.2-kbp genomic fragment from Challis was found to impart spontaneous competence on Wicky by a complementation assay. Wicky clones containing the fragment secreted a heat-sensitive activity that induced competence in Wicky and in a comA insertion mutant of Challis. Activity was localized to a putative open reading frame, comX, with the potential to encode a 52-amino-acid peptide. comX had no similarity to known sequences, and a comX::ermAM insertion mutant of Challis transformed normally and secreted CF. These data suggest that a CF-independent pathway for competence induction exists in S. gordonii.

The zona pellucida is a unique, oocyte-specific matrix that coats the surface of all mammalian eggs. Composed of three sulfated glycoproteins in the mouse (ZP1, ZP2, and ZP3), the zona pellucida facilitates early events in fertilization and protects the embryo during preimplantation development. Using DNA isolated from hamster-mouse somatic cell hybrids and from C57BL/6J X Mus spretus interspecific backcross progeny, Zp-2 was located on chromosome 7, 11.3 +/- 3.2 cM distal to Tyr, and Zp-3 was located on chromosome 5, 9.2 +/- 2.9 cM distal to Gus.

A DNA fragment encoding the sucrose-6-phosphate hydrolase component of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system has been recovered from a plasmid-based genomic library of strain GS5. The locus, designated scrB, was found to reside within a 2.9-kilobase-pair restriction fragment present on the chimeric molecule pVA1343 (7.3 kilobase pairs). Minicell analysis of pVA1343-directed translation products revealed that the scrB product synthesized in Escherichia coli V1343 was a single peptide of Mr 57,000. This polypeptide was reactive with antiserum prepared against S. mutans intracellular invertase, which has been previously shown to have an Mr of 43,000 to 48,000. The basis of this difference in Mr was not established but may represent a posttranslational proteolytic event which occurred in S. mutans but not in recombinant V1343. Sucrose-6-phosphate hydrolase purified to homogeneity from V1343 exhibited Michaelis constants of 180 mM for sucrose and 0.08 mM for sucrose-6-phosphate. Deletion analysis of pVA1343 facilitated the assignment of a coding region for the hydrolase within the insert, as well as an orientation for the transcription of scrB. scrB-defective strains of S. mutans constructed by additive integration of an insertionally inactivated scrB locus exhibited the sucrose sensitivity characteristic of this mutant class. Similar loci were detected by DNA-DNA hybridization in additional strains of S. mutans and two strains of Streptococcus cricetus, but not in single strain representatives of S. rattus, S. sobrinus, S. sanguis I and II, S. salivarius, or S. mitis.

Tn4001 mutagenesis identified a new competence gene in Streptococcus gordonii Challis designated comYA. A comYA mutant was completely deficient in transformation and exhibited decreased levels of DNA binding and hydrolysis. The deduced 319-amino-acid ComYA protein exhibited 57% similarity and 33% identity to the ComGA transporter protein of Bacillus subtilis and contained the Walker A-box motif conserved in ATP-binding proteins as well as aspartic acid boxes Asp-1 and Asp-2 present in some components of the general secretory pathway of gram-negative bacteria. comYA appeared to be part of a putative operon encompassing a comGB homolog, designated comYB, together with sequences that could encode ComGC- and ComGD-like peptides designated ComYC and ComYD, respectively, as well as other components. The putative ComYC and ComYD peptides had leader sequences similar to the type IV N-methylphenylalanine pilins of gram-negative bacteria, but unlike other examples in this class, including B. subtilis, they contained an alanine at position -1 of the leader instead of the usual glycine residue. Northern analysis identified a single 6.0-kb comYA-containing transcript strictly dependent on exogenous competence factor for expression in ComA1 cells. An identical pattern of expression was seen in wild-type Challis cells grown under conditions of maximal competence but not in cells that were noncompetent.

Conditional expression systems were utilized for the ectopic induction of essential genes in Staphylococcus aureus. Resulting strains were then subjected to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. This strategy produced test strains whereby cellular viability was uniquely dependent on the presence of inducer and provided a direct and absolute confirmation of genetic essentiality for each locus. The procedure is particularly useful for genes that are difficult to analyze by conventional inactivation strategies due to either small size or complex genomic organization.

A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.

A strategy based on a vector host-dependent for autonomous replication, pSA3182, was utilized both for the rapid screening for Staphylococcus aureus genes essential for cell viability and for the introduction of specific polarity-neutral deletions in nonessential genes. The results obtained support the use of pSA3182 for both purposes.

The Staphylococcus aureus transposon Tn4001 was found to transpose in Streptococcus gordonii. Transposition sites appeared to be randomly distributed, and the element was stable in the absence of antibiotic selection. An increase in transposition frequency was noted when the delivery plasmid was propagated in a Dam- Dcm- Escherichia coli host strain. The utility of Tn4001 was demonstrated by the generation of lactose-negative mutants. Small size, clonal stability, random transposition, and known nucleotide sequence make Tn4001 a useful addition to the repertoire of tools available to the streptococcal geneticist.

This chapter contains sections titled: Genesis of Human Microbiome Research and the Human Microbiome Project (HMP) Guiding Principles, Structure, and Initiatives of the HMP Program Products from the Human Microbiome Project Other NIH-Supported Human Microbiome Research Activities Future Directions for Human Microbiome Research

Serratia marcescens is an environmental bacterium and clinical pathogen that can cause an array of infections. We describe an environmental sampling and comparative genomics approach used to investigate a multi-year outbreak of S. marcescens at a correctional facility. Whole genome sequencing analysis revealed a predominant cluster of clonally related S. marcescens from nine patient cases and items associated with illicit drug use. Closely related strains found among items associated with case-patient cells and diluted Cell Block 64 (CB64), a quaternary ammonium disinfectant, and Break Out (BO), a multipurpose cleaner, highlighted their role as environmental reservoirs for S. marcescens in this outbreak. Comparative genomic analysis suggested outbreak strains were both persistent (identical strains found over long periods and in multiple locations of the correctional facility) and diverse (strains clustered with multiple global samples from NCBI database). No correlation was found between antimicrobial resistance (AMR) genes of outbreak strains; NCBI strains have more AMR genes. Principal component analysis (PCA) of virulence factors associated with persistence and infectivity indicated variation based on phylogroups, including the predominant cluster; identifiable variations among environmental versus clinical strains were not observed. Identification of multiple distinct genetic groups highlights the importance of putting epidemiological genomic studies in a proper genetic context.

Streptococci are the primary early colonizers in the accretion of dental plaque. Studies have shown that they can adhere to the acquired pellicle of the tooth, to other oral genera, and to other species of oral streptococci9. The surface protein ScaA from Streptococcus gordonii ATCC 51656 has been characterized and is thought to mediate coaggregation of this organism with another early colonizer Actinomyces naeslundii ATCC 51655 (formerly strain PK606)4. ScaA appears as a 38-kDa protein in immunoblots of surface preparations of the wild type strain ATCC 51656, but the protein is absent or greatly reduced in strain PK1804, an isogenic mutant of strain ATCC 51656 which fails to coaggregate with strain ATCC 51655. Immunoblots of surface preparations from eleven other streptococcal coaggregation partners of ATCC 51655 showed the presence of the 38-kDa protein when probed with PK1804-absorbed anti-ATCC 51656 serum1.

Molecular Oral MicrobiologyVolume 25, Issue 1 p. 3-3 Guest editorial R.D. Lunsford, R.D. Lunsford National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USAE-mail: [email protected]Search for more papers by this authorL.A. Tabak, L.A. Tabak National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USAE-mail: [email protected]Search for more papers by this author R.D. Lunsford, R.D. Lunsford National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USAE-mail: [email protected]Search for more papers by this authorL.A. Tabak, L.A. Tabak National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USAE-mail: [email protected]Search for more papers by this author First published: 25 January 2010 https://doi.org/10.1111/j.2041-1014.2009.00557.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume25, Issue1February 2010Pages 3-3 RelatedInformation

The evaluation of the activity of combinations of two or more anti-infective compounds has gained signifi cant prominence in light of the innate ability of many infectious organisms to rapidly acquire drug resistance. Pathogens react to the administration of anti-infective agents by the outgrowth of preexisting infectious clones with resistance-engendering mutations and by the accumulation of new mutations to allow an escape from the suppressive effects of therapeutic drug regimens (1). Resistance emerges through the error-prone mechanisms of the replicative machinery and through the transmission of resistance elements (2, 3), rendering monotherapeutic drug strategies problematic. Combination chemotherapy signifi cantly decreases the risk that resistance will arise. In addition, a combination chemotherapy may ameliorate toxicity by permitting lower, less toxic, or nontoxic concentrations of synergistic drugs to be utilized.

Annals of the New York Academy of SciencesVolume 564, Issue 1 p. 281-288 Developmental Expression of the Sperm Receptor of the Mouse Zona Pellucida JURRIEN DEAN, JURRIEN DEAN Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorMARGARET E. CHAMBERLIN, MARGARET E. CHAMBERLIN Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorSARAH MILLAR, SARAH MILLAR Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorANNE W. BAUR, ANNE W. BAUR Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorR. DWAYNE LUNSFORD, R. DWAYNE LUNSFORD Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this author JURRIEN DEAN, JURRIEN DEAN Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorMARGARET E. CHAMBERLIN, MARGARET E. CHAMBERLIN Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorSARAH MILLAR, SARAH MILLAR Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorANNE W. BAUR, ANNE W. BAUR Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this authorR. DWAYNE LUNSFORD, R. DWAYNE LUNSFORD Laboratory of Cellular and Developmental Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892Search for more papers by this author First published: July 1989 https://doi.org/10.1111/j.1749-6632.1989.tb25905.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume564, Issue1Regulation of Testicular Function Signaling Molecules and Cell‐Cell CommunicationJuly 1989Pages 281-288 RelatedInformation