Cysteine-rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants

Two cysteine-rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58,000 (outer membrane protein 2; OMP2) and 15,000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.     

Differential antimicrobial activity of human mononuclear phagocytes against the human biovars of Chlamydia trachomatis

The antimicrobial activities of human mononuclear phagocytes against Chlamydia trachomatis were investigated. Phagocytes cultured for 7 days or less were efficiently microbicidal. Almost complete inactivation of organisms from both human biovars was observed after 48 hr of incubation. However, organisms from the lymphogranuloma venereum (LGV) biovar survived in mononuclear phagocytes infected after 8 days or more in culture, whereas those from the trachoma biovar continued to be killed by such cells. Phagocytes cultured as long as 21 days killed the trachoma organisms with the same effectiveness as those cultured for 7 days or less. An ultrastructural study of inoculated phagocytes illustrated phagolysosomal fusion with degradation of organisms from either biovar in phagocytes which had been cultured for 24 hr before infection. Phagolysosomal fusion was not observed in cells which had been cultured for 8 days or more and then infected with LGV. The addition of interferon-gamma to these macrophages partially restored the phagocytes' microbicidal activity for LGV. Furthermore, a synergistic effect was observed when eosinophil peroxidase was added with interferon. Specific antibody failed to neutralize the infectivity of LGV organisms in 8-day or older mononuclear phagocytes. The findings may reflect the differences in disease syndromes between the two biovars, with the trachoma biovar causing more peripheral diseases and the LGV biovar causing a more systemic disease, with lymph node involvement as its main syndrome.     

Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development

Twenty one Chlamydia trachomatis reference strains and 40 clinical isolates belonging to the lymphogranuloma venerum (LGV) and trachoma biovars were genotyped by differential restriction mapping of the major-outer-membrane-protein gene (MOMP) obtained by the polymerase-chain reaction (PCR). AluI digestion of the PCR product distinguishes eight MOMP-genotypes corresponding to 8 serovars. Six additional enzymes (NlaIII, CfoI, EcoRI, HinfI, DdeI and FokI) further permit the discrimination of 10 MOMP-genotypes corresponding to the 10 remaining serovars of the species. AluI alone allows direct typing of 78% of the clinical isolates. AluI digestion patterns of mouse C. trachomatis biovar, a C. pneumoniae and two C. psittaci strains, studied for comparison, were clearly distinguishable from one another and from the C. trachomatis LGV and trachoma strains. These results indicate that MOMP genotyping by PCR is a valuable molecular tool for studying C. trachomatis epidemiology.     

Chlamydia trachomatis Mip-like protein

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy. However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs.     

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense"

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.     

In-silico genomic landscape characterization and evolution of SARS-CoV-2 variants isolated in India shows significant drift with high frequency of mutations

In-silico studies on SARS-CoV-2 genome are considered important to identify the significant pattern of variations and its possible effects on the structural and functional characteristics of the virus. The current study determined such genetic variations and their possible impact among SARS-CoV-2 variants isolated in India. A total of 546 SARS-CoV-2 genomic sequences (India) were retrieved from the gene bank (NCBI) and subjected to alignment against the Wuhan variant (NC_045512.2), the corresponding amino acid changes were analyzed using NCBI Protein-BLAST. These 546 variants revealed 841 mutations; most of these were non-synonymous 464/841 (55.1%), there was no identical variant compared to the original strain. All genes; coding and non-coding showed nucleotide changes, most of the structural genes showed frequent nonsynonymous mutations. The most affected genes were ORF1a/b followed by the S gene which showed 515/841 (61.2%) and 120/841 (14.3%) mutations, respectively. The most frequent non-synonymous mutation 486/546 (89.01%) occurred in the S gene (structural gene) at position 23,403 where A changed to G leading to the replacement of aspartic acid by glycine in position (D614G). Interestingly, four variants also showed deletion. The variants MT800923 and MT800925 showed 12 consecutive nucleotide deletion in position 21982–21993 resulting in 4 consecutive amino acid deletions that were leucine, glycine, valine, and tyrosine in positions 141, 142, 143, and 144 respectively. The present study exhibited a higher mutations rate per variant compared to other studies carried out in India.     

The glnB gene of Rhizobium leguminosarum biovar viceae

Abstract An open-reading frame (ORF111) upstream of the glutamine synthetase I structural gene ( glnA ) in Rhizobium leguminosarum biovar viceae encodes a protein which is highly homologous to the P_II protein (encoded by glnB ) of enteric bacteria. ORF111 was cloned in a number of different plasmid vectors and shown to complement a K. pneumoniae glnB mutant. We propose that ORF111 encodes the P_II protein of R. leguminosarum and that it should be designated glnB .     

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28–31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.     

Genetic analysis of bacteriocin 43 of vancomycin-resistant Enterococcus faecium

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.     

Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.     

Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins

Consensus‐sequence engineering has generated protein variants with enhanced stability, and sometimes, with modulated biological function. Consensus mutations are often interpreted as the introduction of ancestral amino acid residues. However, the precise relationship between consensus engineering and ancestral protein resurrection is not fully understood. Here, we report the properties of proteins encoded by consensus sequences derived from a multiple sequence alignment of extant, class A β‐lactamases, as compared with the properties of ancient Precambrian β‐lactamases resurrected in the laboratory. These comparisons considered primary sequence, secondary, and tertiary structure, as well as stability and catalysis against different antibiotics. Out of the three consensus variants generated, one could not be expressed and purified (likely due to misfolding and/or low stability) and only one displayed substantial stability having substrate promiscuity, although to a lower extent than ancient β‐lactamases. These results: (i) highlight the phenotypic differences between consensus variants and laboratory resurrections of ancestral proteins; (ii) question interpretations of consensus proteins as phenotypic proxies of ancestral proteins; and (iii) support the notion that ancient proteins provide a robust approach toward the preparation of protein variants having large numbers of mutational changes while possessing unique biomolecular properties. Proteins 2014; 82:887–896. © 2014 Wiley Periodicals, Inc.     

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57.

A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.     

猪圆环病毒2型ORF2基因序列分析及真核表达载体的构建

根据ＧｅｎＢａｎｋ中猪圆环病毒２型（ＰＣＶ－２）ＯＲＦ２基因序列,设计一　对引物,应用ＰＣＲ从疑患断奶仔猪多系统消耗综合症（ＰＭＷＳ）的死亡仔猪组织病料中扩增出ＯＲＦ　２全基因（７０２ｂｐ）。将此片段克隆入ｐＧＥＭ－Ｔ　ｅａｓｙ载体,筛选获得重组质粒ｐＴＯＲＦ２,并对此质　粒中的插入序列进行了测序分析,结果表明本试验克隆的ＯＲＦ２与美国ＰＣＶ－２分离株ＡＦ２６４０３９　的核苷酸及氨基酸序列同源性均达到１００％,与其他ＰＣＶ－２毒株同源性分别为９２．３％～９８．　６％和　９２．３％～９６．６％。重组质粒ｐＴＯＲＦ２经　Ｂａｍ　Ｈ　Ｉ、Ｅｃｏ　Ｒ　Ｖ双酶切,回收ＯＲＦ２基因,转　移入真　核表达载体ｐＳｅｃＴａｇ２／ＨｙｇｒｏＢ的相应酶切位点之间,构建成重组质粒ｐＳｅｃＴａｇＯＲＦ２。此重组表　达载体的构建成功为进一步研究ＯＲＦ２编码蛋白的生物学活性及建立ＰＣＶ诊断试剂盒打下基础。     

中华蜜蜂囊状幼虫病毒北京分离株VP1蛋白基因序列特征及原核表达

【目的】研究中华蜜蜂囊状幼虫病毒（Chinese sacbrood virus, CSBV）VP1蛋白的分子进化特征及遗传多样性。【方法】利用RT-PCR方法,克隆了8株CSBV北京分离株VP1蛋白的基因编码区。【结果】序列分析表明,VP1蛋白基因编码区开放阅读框长945 bp,编码315个氨基酸,推测编码蛋白的相对分子量和等电点分别为35.42 kDa和9.23,具有亲水性和免疫原性。序列同源性分析表明,不同年份CSBV北京分离株VP1蛋白氨基酸序列间差异较小,仅个别氨基酸存在差异。北京分离株与辽宁分离株及越南分离株VP1核苷酸序列一致性达93%,与印度及韩国分离株VP1核苷酸序列一致性达92%,与英国分离株VP1核苷酸序列一致性最低,为88%。序列分析同时表明,CSBV北京分离株VP1蛋白序列存在特有的序列特征,同其他地区分离株比较,北京分离株VP1蛋白序列中存在着氨基酸的插入突变。序列替换率分析表明,亚洲型分离株间序列替换率低于亚洲分离株与欧洲分离株间的替换率。构建原核表达载体pEASY-E1-VP1,经IPTG诱导,CSBV VP1蛋白在大肠杆菌Escherichia coli BL21（DE3）pLysS菌株中表达。【结论】本研究提示CSBV不同分离株基因序列存在变异,结果为进一步研究CSBV致病性分化的分子机理奠定了基础。     

Neurovirulence in mice of H5N1 influenza virus genotypes isolated from Hong Kong poultry in 2001

We studied the pathogenicity of five different genotypes (A to E) of highly pathogenic avian H5N1 viruses, which contained HA genes similar to those of the H5N1 virus A/goose/Guangdong/1/96 and five different combinations of "internal" genes, in a mouse model. Highly pathogenic, neurotropic variants of genotypes A, C, D, and E were isolated from the brain after a single intranasal passage in mice. Genotype B virus was isolated from lungs only. The mouse brain variants had amino acid changes in all gene products except PB1, NP, and NS1 proteins but no common sets of mutations. We conclude that the original H5N1/01 isolates of genotypes A, C, D, and E were heterogeneous and that highly pathogenic neurotropic variants can be rapidly selected in mice.     

Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.