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1.  Evaluation and selection of tandem repeat loci for Streptococcus pneumoniae MLVA strain typing 
BMC Microbiology  2005;5:66.
Precise identification of bacterial pathogens at the strain level is essential for epidemiological purposes. In Streptococcus pneumoniae, the existence of 90 different serotypes makes the typing particularly difficult and requires the use of highly informative tools. Available methods are relatively expensive and cannot be used for large-scale or routine typing of any new isolate. We explore here the potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats), a method of growing importance in the field of molecular epidemiology, for genotyping of Streptococcus pneumoniae.
Available genome sequences were searched for polymorphic tandem repeats. The loci identified were typed across a collection of 56 diverse isolates and including a group of serotype 1 isolates from Africa. Eventually a set of 16 VNTRs was proposed for MLVA-typing of S. pneumoniae. These robust markers were sufficient to discriminate 49 genotypes and to aggregate strains on the basis of the serotype and geographical origin, although some exceptions were found. Such exceptions may reflect serotype switching or horizontal transfer of genetic material.
We describe a simple PCR-based MLVA genotyping scheme for S. pneumoniae which may prove to be a powerful complement to existing tools for epidemiological studies. Using this technique we uncovered a clonal population of strains, responsible for infections in Burkina Faso. We believe that the proposed MLVA typing scheme can become a standard for epidemiological studies of S. pneumoniae.
PMCID: PMC1315331  PMID: 16287512
2.  Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR 
BMC Microbiology  2005;5:65.
Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed.
We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background.
The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.
PMCID: PMC1298302  PMID: 16280082
3.  The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora 
BMC Microbiology  2005;5:64.
The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog.
Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Δtap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains.
The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.
PMCID: PMC1298301  PMID: 16266439
4.  M protein typing of Thai group A streptococcal isolates by PCR-Restriction fragment length polymorphism analysis 
BMC Microbiology  2005;5:63.
Group A streptococcal (GAS) infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF) and rheumatic heart disease (RHD). RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT) by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assay for molecular typing the M protein gene (emm) of GAS.
Using one pair of primers, 13 known GAS M types showed one to four bands of PCR products and after digestion with Alu I, they gave different RFLP patterns. Of 106 GAS isolates examined from the normal Thai population and from patients with GAS-associated complications including RHD, 95 isolates gave RFLP patterns that corresponded to the 13 known M types. Only 11 isolates gave RFLP patterns that differed from the 13 known M types. These were then analyzed by DNA sequencing and six additional M types were identified. In addition, we found that M93 GAS was the most common M type in the population studied, and is consistent with a previous study of Thai GAS isolates.
PCR-RFLP analysis has the potential for the rapid screening of different GAS M types and is therefore considerably advantageous as an alternative M typing approach in developing countries in which GAS is endemic.
PMCID: PMC1274321  PMID: 16225702
5.  Comparison between Gram stain and culture for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora 
BMC Microbiology  2005;5:61.
The microbiological diagnosis of bacterial vaginosis is usually made using Nugent's criteria, a useful but rather laborious scoring system based on counting bacterial cell types on Gram stained slides of vaginal smears. Ison and Hay have simplified the score system to three categories and added a fourth category for microflora with a predominance of the Streptococcus cell type. Because in the Nugent system several cell types are not taken into account for a final score, we carried out a detailed assessment of the composition of the vaginal microflora in relation to standard Gram stain in order the improve the diagnostic value of the Gram stain. To this purpose we compared Gram stain based categorization of vaginal smears with i) species specific PCR for the detection of Gardnerella vaginalis and Atopobium vaginae and with ii) tDNA-PCR for the identification of most cultivable species.
A total of 515 samples were obtained from 197 pregnant women, of which 403 (78.3%) were categorized as grade I microflora, 46 (8.9%) as grade II, 22 (4.3%) as grade III and 8 (1.6%) as grade IV, according to the criteria of Ison and Hay. Another 36 samples (7.0%) were assigned to the new category 'grade I-like', because of the presence of diphtheroid bacilli cell types. We found that 52.7% of the grade I-like samples contained Bifidobacterium spp. while L. crispatus was present in only 2.8% of the samples and G. vaginalis and A. vaginae were virtually absent; in addition, the species diversity of this category was similar to that of grade II specimens.
Based on the presence of different Lactobacillus cell types, grade I specimens were further characterized as grade Ia (40.2%), grade Iab (14.9%) and grade Ib (44.9%). We found that this classification was supported by the finding that L. crispatus was cultured from respectively 87.0% and 76.7% of grade Ia and Iab specimens while this species was present in only 13.3% of grade Ib specimens, a category in which L. gasseri and L. iners were predominant.
Further refinement of Gram stain based grading of vaginal smears is possible by distinguishing additional classes within grade I smears (Ia, Iab and Ib) and by adding a separate category, designated grade I-like. A strong correlation was found between grade Ia and the presence of L. crispatus and between grade I-like and the presence of bifidobacteria. This refinement of Gram stain based scoring of vaginal smears may be helpful to improve the interpretation of the clinical data in future studies, such as the understanding of response to treatment and recurrence of bacterial vaginosis in some women, and the relationship between bacterial vaginosis and preterm birth.
PMCID: PMC1266370  PMID: 16225680
6.  ABC transporter FtsABCD of Streptococcus pyogenes mediates uptake of ferric ferrichrome 
BMC Microbiology  2005;5:62.
The Streptococcus pyogenes or Group A Streptococcus (GAS) genome encodes three ABC transporters, namely, FtsABCD, MtsABC, and HtsABC, which share homology with iron transporters. MtsABC and HtsABC are believed to take up ferric (Fe3+) and manganese ions and heme, respectively, while the specificity of FtsABCD is unknown.
Recombinant FtsB, the lipoprotein component of FtsABCD, was found to bind Fe3+ ferrichrome in a 1:1 stoichiometry. To investigate whether FtsABCD transports Fe3+ ferrichrome, GAS isogenic strains defective in lipoprotein gene ftsB and permease gene ftsC were generated, and the effects of the mutations on uptake of Fe3+ ferrichrome were examined using radioactive 55Fe3+ ferrichrome. FtsB was produced in the wild-type strain but not in the ftsB mutant, confirming the ftsB inactivation. While wild-type GAS took up 3.6 × 104 Fe3+ ferrichrome molecules per bacterium per min at room temperature, the ftsB and ftsC mutants did not have a detectable rate of Fe3+ ferrichrome uptake. The inactivation of ftsB or ftsC also decreased 55Fe3+ ferrichrome uptake by >90% under growth conditions in the case of limited uptake time. Complementation of the ftsB mutant with a plasmid carrying the ftsB gene restored FtsB production and 55Fe3+ ferrichrome association at higher levels compared with the parent strain. The inactivation of mtsA and htsA and Fe-restricted conditions enhanced the production of FtsB and Fe3+ ferrichrome uptake.
The FtsB protein bound Fe3+ ferrichrome, and inactivation of ftsB or ftsC, but not htsA or mtsA, diminished Fe3+ ferrichrome uptake, indicating that FtsABCD, but not HtsABC and MtsABC, is the transporter that takes up Fe3+ ferrichrome in GAS. Fe acquisition systems are virulence factors in many bacterial pathogens and are attractive vaccine candidates. The elucidation of the FtsABCD specificity advances the understanding of Fe acquisition processes in GAS and may help evaluating the GAS Fe acquisition systems as vaccine candidates.
PMCID: PMC1276799  PMID: 16225685
7.  Genometrics as an essential tool for the assembly of whole genome sequences: the example of the chromosome of Bifidobacterium longum NCC2705 
BMC Microbiology  2005;5:60.
Analysis of the first reported complete genome sequence of Bifidobacterium longum NCC2705, an actinobacterium colonizing the gastrointestinal tract, uncovered its proteomic relatedness to Streptomyces coelicolor and Mycobacterium tuberculosis. However, a rapid scrutiny by genometric methods revealed a genome organization totally different from all so far sequenced high-GC Gram-positive chromosomes.
Generally, the cumulative GC- and ORF orientation skew curves of prokaryotic genomes consist of two linear segments of opposite slope: the minimum and the maximum of the curves correspond to the origin and the terminus of chromosome replication, respectively. However, analyses of the B. longum NCC2705 chromosome yielded six, instead of two, linear segments, while its dnaA locus, usually associated with the origin of replication, was not located at the minimum of the curves. Furthermore, the coorientation of gene transcription with replication was very low.
Comparison with closely related actinobacteria strongly suggested that the chromosome of B. longum was misassembled, and the identification of two pairs of relatively long homologous DNA sequences offers the possibility for an alternative genome assembly proposed here below. By genometric criteria, this configuration displays all of the characters common to bacteria, in particular to related high-GC Gram-positives. In addition, it is compatible with the partially sequenced genome of DJO10A B. longum strain. Recently, a corrected sequence of B. longum NCC2705, with a configuration similar to the one proposed here below, has been deposited in GenBank, confirming our predictions.
Genometric analyses, in conjunction with standard bioinformatic tools and knowledge of bacterial chromosome architecture, represent fast and straightforward methods for the evaluation of chromosome assembly.
PMCID: PMC1285363  PMID: 16223444
8.  Polyamine stress at high pH in Escherichia coli K-12 
BMC Microbiology  2005;5:59.
Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress.
The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range). At pH 2, 10 mM spermine increased survival by 2-fold, and putrescine increased survival by 30%. At pH 9.8, however, E. coli survival was decreased 100-fold by 10 mM spermine, putrescine, cadaverine, or spermidine. At pH 8.5, spermine decreased the growth rate substantially, whereas little effect was seen at pH 5.5. Spermidine required ten-fold higher concentrations to impair growth. On proteomic 2-D gels, spermine and spermidine caused differential expression of 31 different proteins. During log-phase growth at pH 7.0, 1 mM spermine induced eight proteins, including PykF, GlpK, SerS, DeaD, OmpC and OmpF. Proteins repressed included acetate-inducible enzymes (YfiD, Pta, Lpd) as well as RapA (HepA), and FabB. At pH 8.5, spermine induced additional proteins: TnaA, OmpA, YrdA and NanA (YhcJ) and also repressed 17 proteins. Four of the proteins that spermine induced (GlpK, OmpA, OmpF, TnaA) and five that were repressed (Lpd, Pta, SucB, TpiA, YfiD) show similar induction or repression, respectively, in base compared to acid. Most of these base stress proteins were also regulated by spermidine, but only at ten-fold higher concentration (10 mM) at high pH (pH 8.5).
Polyamines increase survival in extreme acid, but decrease E. coli survival in extreme base. Growth inhibition by spermine and spermidine requires neutral or higher pH. At or above pH 7, spermine and spermidine regulate specific proteins, many of which are known to be regulated by base stress. High pH amplifies polyamine stress; and naturally occurring polyamines may play an important role in base stress.
PMCID: PMC1274320  PMID: 16223443
9.  Non-classical protein secretion in bacteria 
BMC Microbiology  2005;5:58.
We present an overview of bacterial non-classical secretion and a prediction method for identification of proteins following signal peptide independent secretion pathways. We have compiled a list of proteins found extracellularly despite the absence of a signal peptide. Some of these proteins also have known roles in the cytoplasm, which means they could be so-called "moon-lightning" proteins having more than one function.
A thorough literature search was conducted to compile a list of currently known bacterial non-classically secreted proteins. Pattern finding methods were applied to the sequences in order to identify putative signal sequences or motifs responsible for their secretion. We have found no signal or motif characteristic to any majority of the proteins in the compiled list of non-classically secreted proteins, and conclude that these proteins, indeed, seem to be secreted in a novel fashion. However, we also show that the apparently non-classically secreted proteins are still distinguished from cellular proteins by properties such as amino acid composition, secondary structure and disordered regions. Specifically, prediction of disorder reveals that bacterial secretory proteins are more structurally disordered than their cytoplasmic counterparts. Finally, artificial neural networks were used to construct protein feature based methods for identification of non-classically secreted proteins in both Gram-positive and Gram-negative bacteria.
We present a publicly available prediction method capable of discriminating between this group of proteins and other proteins, thus allowing for the identification of novel non-classically secreted proteins. We suggest candidates for non-classically secreted proteins in Escherichia coli and Bacillus subtilis. The prediction method is available online.
PMCID: PMC1266369  PMID: 16212653
10.  The two authentic methionine aminopeptidase genes are differentially expressed in Bacillus subtilis 
BMC Microbiology  2005;5:57.
Two putative methionine aminopeptidase genes, map (essential) and yflG (non-essential), were identified in the genome sequence of Bacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in B. subtilis.
In silico analysis of MAP evolution uncovered a coordinated pattern of MAP and deformylase that did not correlate with the pattern of 16S RNA evolution. Biochemical assays showed that both MAP (MAP_Bs) and YflG (YflG_Bs) from B. subtilis overproduced in Escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. Compared with MAP_Bs, YflG_Bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates. Both map and yflG genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both E. coli and B. subtilis. In contrast, lacZ gene transcriptional fusions showed that the promoter activity of map was 50 to 100-fold higher than that of yflG. Primer extension analysis detected the transcription start site of the yflG promoter. Further work identified that YvoA acted as a possible weak repressor of yflG expression in B. subtilis in vivo.
Both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vitro and in vivo. The high expression level of map and low expression level of yflG may account for their essentiality and dispensality in B. subtilis, respectively, when cells are grown under laboratory conditions. Their difference in activity on synthetic substrates suggests that they have different protein targets in vivo.
PMCID: PMC1266368  PMID: 16207374
11.  A gonococcal homologue of meningococcal γ-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent 
BMC Microbiology  2005;5:56.
It has been speculated that the γ-glutamyl transpeptidase (ggt) gene is present only in Neisseria meningitidis and not among related species such as Neisseria gonorrhoeae and Neisseria lactamica, because N. meningitidis is the only bacterium with GGT activity. However, nucleotide sequences highly homologous to the meningococcal ggt gene were found in the genomes of N. gonorrhoeae isolates.
The gonococcal homologue (ggt gonococcal homologue; ggh) was analyzed. The nucleotide sequence of the ggh gene was approximately 95 % identical to that of the meningococcal ggt gene. An open reading frame in the ggh gene was disrupted by an ochre mutation and frameshift mutations induced by a 7-base deletion, but the amino acid sequences deduced from the artificially corrected ggh nucleotide sequences were approximately 97 % identical to that of the meningococcal ggt gene. The analyses of the sequences flanking the ggt and ggh genes revealed that both genes were localized in a common DNA region containing the fbp-ggt (or ggh)-glyA-opcA-dedA-abcZ gene cluster. The expression of the ggh RNA could be detected by dot blot, RT-PCR and primer extension analyses. Moreover, the truncated form of ggh-translational product was also found in some of the gonococcal isolates.
This study has shown that the gonococcal ggh gene is a pseudogene of the meningococcal ggt gene, which can also be designated as Ψggt. The gonococcal ggh (Ψggt) gene is the first identified bacterial pseudogene that is transcriptionally active but phenotypically silent.
PMCID: PMC1262726  PMID: 16202144
12.  High-throughput metal susceptibility testing of microbial biofilms 
BMC Microbiology  2005;5:53.
Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated.
This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO32-) than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic cell E. coli JM109 to metals was time-dependent.
This method results in accurate, easily reproducible comparisons between the susceptibility of planktonic cells and biofilms to metals. Further, it was possible to make direct comparisons of the ability of different microbial strains to withstand metal toxicity. The data presented here also indicate that exposure time is an important variable in metal susceptibility testing of bacteria.
PMCID: PMC1262724  PMID: 16202124
13.  Comparative analysis of RNA regulatory elements of amino acid metabolism genes in Actinobacteria 
BMC Microbiology  2005;5:54.
Formation of alternative structures in mRNA in response to external stimuli, either direct or mediated by proteins or other RNAs, is a major mechanism of regulation of gene expression in bacteria. This mechanism has been studied in detail using experimental and computational approaches in proteobacteria and Firmicutes, but not in other groups of bacteria.
Comparative analysis of amino acid biosynthesis operons in Actinobacteria resulted in identification of conserved regions upstream of several operons. Classical attenuators were predicted upstream of trp operons in Corynebacterium spp. and Streptomyces spp., and trpS and leuS genes in some Streptomyces spp. Candidate leader peptides with terminators were observed upstream of ilvB genes in Corynebacterium spp., Mycobacterium spp. and Streptomyces spp. Candidate leader peptides without obvious terminators were found upstream of cys operons in Mycobacterium spp. and several other species. A conserved pseudoknot (named LEU element) was identified upstream of leuA operons in most Actinobacteria. Finally, T-boxes likely involved in the regulation of translation initiation were observed upstream of ileS genes from several Actinobacteria.
The metabolism of tryptophan, cysteine and leucine in Actinobacteria seems to be regulated on the RNA level. In some cases the mechanism is classical attenuation, but in many cases some components of attenuators are missing. The most interesting case seems to be the leuA operon preceded by the LEU element that may fold into a conserved pseudoknot or an alternative structure. A LEU element has been observed in a transposase gene from Bifidobacterium longum, but it is not conserved in genes encoding closely related transposases despite a very high level of protein similarity. One possibility is that the regulatory region of the leuA has been co-opted from some element involved in transposition. Analysis of phylogenetic patterns allowed for identification of ML1624 of M. leprae and its orthologs as the candidate regulatory proteins that may bind to the LEU element. T-boxes upstream of the ileS genes are unusual, as their regulatory mechanism seems to be inhibition of translation initiation via a hairpin sequestering the Shine-Dalgarno box.
PMCID: PMC1262725  PMID: 16202131
14.  In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta-analysis and meta-regression 
BMC Microbiology  2005;5:55.
More than 200 studies related to nucleic acid amplification (NAA) tests to detect Mycobacterium tuberculosis directly from clinical specimens have appeared in the world literature since this technology was first introduced. NAA tests come as either commercial kits or as tests designed by the reporting investigators themselves (in-house tests). In-house tests vary widely in their accuracy, and factors that contribute to heterogeneity in test accuracy are not well characterized. Here, we used meta-analytical methods, including meta-regression, to identify factors related to study design and assay protocols that affect test accuracy in order to identify those factors associated with high estimates of accuracy.
By searching multiple databases and sources, we identified 2520 potentially relevant citations, and analyzed 84 separate studies from 65 publications that dealt with in-house NAA tests to detect M. tuberculosis in sputum samples. Sources of heterogeneity in test accuracy estimates were determined by subgroup and meta-regression analyses. Among 84 studies analyzed, the sensitivity and specificity estimates varied widely; sensitivity varied from 9.4% to 100%, and specificity estimates ranged from 5.6% to 100%. In the meta-regression analysis, the use of IS6110 as a target, and the use of nested PCR methods appeared to be significantly associated with higher diagnostic accuracy.
Estimates of accuracy of in-house NAA tests for tuberculosis are highly heterogeneous. The use of IS6110 as an amplification target, and the use of nested PCR methods appeared to be associated with higher diagnostic accuracy. However, the substantial heterogeneity in both sensitivity and specificity of the in-house NAA tests rendered clinically useful estimates of test accuracy difficult. Future development of NAA-based tests to detect M. tuberculosis from sputum specimens should take into consideration these findings in improving accuracy of in-house NAA tests.
PMCID: PMC1260021  PMID: 16202138
15.  A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results 
BMC Microbiology  2005;5:52.
Bacillus thuringiensis is a bacterium known for producing protein crystals with insecticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. There is great interest in isolating strains with improved or novel toxin characteristics, however isolating B. thuringiensis from the environment is time consuming and yields relatively few isolates of interest. New approaches to B. thuringiensis isolation have been, and continue to be sought. In this report, candidate B. thuringiensis isolates were recovered from environmental samples using a combination of a novel stain, high throughput and reduced selection. Isolates were further characterized by SDS-PAGE, light microscopy, PCR, probe hybridization, and with selected isolates, DNA sequencing, bioassay or Electron Microscopy.
Based on SDS-PAGE patterns and the presence of cry genes or a crystal, 79 candidate, non-clonal isolates of B. thuringiensis were identified from 84 samples and over 10,000 colonies. Although only 16/79 (20%) of the isolates showed DNA homology by Probe Hybridization or PCR to common cry genes, initial characterization revealed a surprisingly rich library that included a putative nematocidal gene, a novel filamentous structure associated with a crystal, a spore with spikes originating from a very small parasporal body and isolates with unusually small crystals. When compared to reports of other screens, this screen was also atypical in that only 3/79 isolates (3.8%) produced a bipyramidal crystal and 24/79 (30%) of the isolates' spores possessed an attached, dark-staining body.
Results suggest that the screening methodology adopted in this study might deliver a vastly richer and potentially more useful library of B. thuringiensis isolates as compared to that obtained with commonly reported methodologies, and that by extension, methodologies fundamentally different from current methods should also be explored.
PMCID: PMC1253514  PMID: 16181492
16.  Mycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves 
BMC Microbiology  2005;5:51.
The current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface.
The work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases.
An early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.
PMCID: PMC1249576  PMID: 16164744
17.  Expression capable library for studies of Neisseria gonorrhoeae, version 1.0 
BMC Microbiology  2005;5:50.
The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system.
The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci.
This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.
PMCID: PMC1236931  PMID: 16137322
18.  Identification of a new family of enzymes with potential O-acetylpeptidoglycan esterase activity in both Gram-positive and Gram-negative bacteria 
BMC Microbiology  2005;5:49.
The metabolism of the rigid bacterial cell wall heteropolymer peptidoglycan is a dynamic process requiring continuous biosynthesis and maintenance involving the coordination of both lytic and synthetic enzymes. The O-acetylation of peptidoglycan has been proposed to provide one level of control on these activities as this modification inhibits the action of the major endogenous lytic enzymes, the lytic transglycosylases. The O-acetylation of peptidoglycan also inhibits the activity of the lysozymes which serve as the first line of defense of host cells against the invasion of bacterial pathogens. Despite this central importance, there is a dearth of information regarding peptidoglycan O-acetylation and nothing has previously been reported on its de-acetylation.
Homology searches of the genome databases have permitted this first report on the identification of a potential family of O-Acetylpeptidoglycan esterases (Ape). These proteins encoded in the genomes of a variety of both Gram-negative and Gram-positive bacteria, including a number of important human pathogens such as species of Neisseria, Helicobacter, Campylobacter, and Bacillus anthracis, have been organized into three families based on amino acid sequence similarities with family 1 being further divided into three sub-families. The genes encoding these proteins are shown to be clustered with Peptidoglycan O-acetyltransferases (Pat) and in some cases, together with other genes involved in cell wall metabolism. Representative bacteria that encode the Ape proteins were experimentally shown to produce O-acetylated peptidoglycan.
The hypothetical proteins encoded by the pat and ape genes have been organized into families based on sequence similarities. The Pat proteins have sequence similarity to Pseudomonas aeruginosa AlgI, an integral membrane protein known to participate in the O-acetylation of the exopolysaccaride, alginate. As none of the bacteria that harbor the pat genes produce alginate, we propose that the Pat proteins serve to O-acetylate peptidoglycan which is known to be a maturation event occurring in the periplasm. The Ape sequences have amino acid sequence similarity to the CAZy CE 3 carbohydrate esterases, a family previously known to be composed of only O-acetylxylan esterases. They are predicted to contain the α/β hydrolase fold associated with the GDSL and TesA hydrolases and they possess the signature motifs associated with the catalytic residues of the CE3 esterases. Specific signature sequence motifs were identified for the Ape proteins which led to their organization into distinct families. We propose that by expressing both Pat and Ape enzymes, bacteria would be able to obtain a high level of localized control over the degradation of peptidoglycan through the attachment and removal of O-linked acetate. This would facilitate the efficient insertion of pores and flagella, localize spore formation, and control the level of general peptidoglycan turnover.
PMCID: PMC1199599  PMID: 16111493
19.  Herpes simplex virus interferes with amyloid precursor protein processing 
BMC Microbiology  2005;5:48.
The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-ε4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39–43 amino acid protein – β amyloid (Aβ) – within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Aβ or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Aβ; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting.
We have found that acute HSV1 (and also HSV2) infection rapidly reduces full length APP levels – as might be expected – yet surprisingly markedly increases levels of a novel C-terminal fragment of APP of about 55 kDa. This band was not increased in cells treated with the protein synthesis inhibitor cycloheximide
Herpes virus infection leads to rapid loss of full length APP from cells, yet also causes increased levels of a novel 55 kDa C-terminal APP fragment. These data suggest that infection can directly alter the processing of a transmembranal protein intimately linked to the aetiology of AD.
PMCID: PMC1198230  PMID: 16109164
20.  Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors 
BMC Microbiology  2005;5:46.
The oomycete Saprolegnia parasitica is one of the most economically important fish pathogens. There is a dramatic recrudescence of Saprolegnia infections in aquaculture since the use of the toxic organic dye malachite green was banned in 2002. Little is known about the molecular mechanisms underlying pathogenicity in S. parasitica and other animal pathogenic oomycetes. In this study we used a genomics approach to gain a first insight into the transcriptome of S. parasitica.
We generated 1510 expressed sequence tags (ESTs) from a mycelial cDNA library of S. parasitica. A total of 1279 consensus sequences corresponding to 525944 base pairs were assembled. About half of the unigenes showed similarities to known protein sequences or motifs. The S. parasitica sequences tended to be relatively divergent from Phytophthora sequences. Based on the sequence alignments of 18 conserved proteins, the average amino acid identity between S. parasitica and three Phytophthora species was 77% compared to 93% within Phytophthora. Several S. parasitica cDNAs, such as those with similarity to fungal type I cellulose binding domain proteins, PAN/Apple module proteins, glycosyl hydrolases, proteases, as well as serine and cysteine protease inhibitors, were predicted to encode secreted proteins that could function in virulence. Some of these cDNAs were more similar to fungal proteins than to other eukaryotic proteins confirming that oomycetes and fungi share some virulence components despite their evolutionary distance
We provide a first glimpse into the gene content of S. parasitica, a reemerging oomycete fish pathogen. These resources will greatly accelerate research on this important pathogen. The data is available online through the Oomycete Genomics Database [1].
PMCID: PMC1192801  PMID: 16076392
21.  The influence of acetyl phosphate on DspA signalling in the Cyanobacterium Synechocystis sp. PCC6803 
BMC Microbiology  2005;5:47.
The dspA (hik33) gene, coding for a putative sensory histidine kinase, is conserved in plastids (ycf26) and cyanobacteria. It has been linked with a number of different stress responses in cyanobacteria.
We constructed an insertional mutant of dspA (ycf26) in Synechocystis 6803. We found little phenotypic effect during nitrogen starvation. However, when the mutation was combined with deletion of the pta gene coding for phosphotransacetylase, a more significant phenotype was observed. Under nitrogen starvation, the pta/dspA double mutant degrades its phycobilisomes less than the wild type and still has about half of its chlorophyll-protein complexes.
Our data indicates that acetyl-phosphate-dependent phosphorylation of response regulator(s) overlaps with DspA-dependent signalling of the degradation of chlorophyll-protein complexes (and to a lesser extent phycobilisomes) in Synechocystis 6803.
PMCID: PMC1192802  PMID: 16076400
22.  Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein 
BMC Microbiology  2005;5:45.
Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.
The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals.
Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines.
PMCID: PMC1187892  PMID: 16076390
23.  Genotyping of Mycobacterium tuberculosis clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor 
BMC Microbiology  2005;5:44.
Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia.
A total of 245 DNAs from Mycobacterium tuberculosis clinical isolates from tuberculosis patients residing in Turkey (Malatya n = 147 or Ankara n = 98) were genotyped by spoligotyping, a high-throughput genotyping method based on the polymorphism of the Direct Repeat locus. Thirty-three spoligotyping-defined clusters including 206 patients and 39 unique patterns were found. The ST41 cluster, as designated according to the international SpolDB3 database project, represented one fourth and when gathered to three genotypes, ST53, ST50 and ST284, one half of all the isolates. Out of 34 clinical isolates harboring ST41 which were further genotyped by IS6110 and by MIRU-VNTR typing, a typical 2-copy IS6110-RFLP pattern and a "215125113322" MIRU-VNTR pattern were observed among 21 clinical isolates. Further search in various databases confirms the likely Turkish-phylogeographical specificity of this clonal complex.
We described a new phylogeographically-specific clone of M. tuberculosis, designated LAM7-TUR. Further investigations to assess its frequency within all regions of Turkey and its phylogeographical origin and phylogenetic position within the global M. tuberculosis phylogenetic tree will shed new light on its endemicity in Asia Minor.
PMCID: PMC1192800  PMID: 16045794
24.  Characterization of Pseudomonas aeruginosa isolated from chronically infected children with cystic fibrosis in India 
BMC Microbiology  2005;5:43.
Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism resides as a biofilm, shows multi-drug resistance, diversifies its colony morphology and becomes auxotrophic. The patients have been found to be colonized with multiple genotypes. The present work was carried out to characterize P. aeruginosa isolated from children with cystic fibrosis using phenotypic and genotypic methods.
We studied 56 patients with CF attending the Pediatric Chest clinic at All India Institute of Medical Sciences, New Delhi, India during August 1998-August 2001. These patients were regularly followed up at the clinic. Out of 56 patients, 27 were culture positive for P. aeruginosa where 8 were chronically infected (Group1) and 19 were intermittently colonized with the organism (Group2). Patients under Group1 had significantly higher rates of hospitalization, death and colonization with different colony morphotypes (p < 0.05). The isolates from Group1 patients were the positive producers of extended spectrum beta lactamase. A total of 5 auxotrophs were recovered from 2 patients where one was chronically infected with P. aeruginosa and the other was a recently enrolled patient. The auxotrophs had the specific requirement for methionine and arginine. Molecular typing revealed 33 ERIC-PCR (E1-E33) and 5 PCR-ribotyping (P1-P5) patterns. By ERIC-PCR, 4 patients were colonized with 2–4 genotypes and the remaining 23 patients were colonized with the single genotype.
With chronicity of infection, P. aeruginosa becomes multidrug resistant, diversifies its colony morphology, acquires mucoidity and shows auxotrophy for amino acids. The chronically infected patients can be colonized with multiple genotypes. Thus in a particular clinical set up, high index of suspicion should be there for diagnosis of CF patients so as to prevent the delay in diagnosis and management of CF patients.
PMCID: PMC1183212  PMID: 16033658
25.  Proteome analysis of serovars Typhimurium and Pullorum of Salmonella enterica subspecies I 
BMC Microbiology  2005;5:42.
Salmonella enterica subspecies I includes several closely related serovars which differ in host ranges and ability to cause disease. The basis for the diversity in host range and pathogenic potential of the serovars is not well understood, and it is not known how host-restricted variants appeared and what factors were lost or acquired during adaptations to a specific environment. Differences apparent from the genomic data do not necessarily correspond to functional proteins and more importantly differential regulation of otherwise identical gene content may play a role in the diverse phenotypes of the serovars of Salmonella.
In this study a comparative analysis of the cytosolic proteins of serovars Typhimurium and Pullorum was performed using two-dimensional gel electrophoresis and the proteins of interest were identified using mass spectrometry. An annotated reference map was created for serovar Typhimurium containing 233 entries, which included many metabolic enzymes, ribosomal proteins, chaperones and many other proteins characteristic for the growing cell. The comparative analysis of the two serovars revealed a high degree of variation amongst isolates obtained from different sources and, in some cases, the variation was greater between isolates of the same serovar than between isolates with different sero-specificity. However, several serovar-specific proteins, including intermediates in sulphate utilisation and cysteine synthesis, were also found despite the fact that the genes encoding those proteins are present in the genomes of both serovars.
Current microbial proteomics are generally based on the use of a single reference or type strain of a species. This study has shown the importance of incorporating a large number of strains of a species, as the diversity of the proteome in the microbial population appears to be significantly greater than expected. The characterisation of a diverse selection of strains revealed parts of the proteome of S. enterica that alter their expression while others remain stable and allowed for the identification of serovar-specific factors that have so far remained undetected by other methods.
PMCID: PMC1181816  PMID: 16026608

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