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In vivo analysis of puf operon expression in Rhodobacter sphaeroides after deletion of a putative intercistronic transcription terminator.

The intercistronic region of the mRNA derived from the puf operon of Rhodobacter sphaeroides is capable of forming two stable stem-loop structures, the first of which resembles a factor-independent transcription terminator. A puf operon construction lacking the putative transcription terminator was made in vitro and crossed into the chromosome of R. sphaeroides PUFB1 to yield a single chromosomal copy in the terminator-deleted strain. The mutant strain, designated PUF delta 348-420 which was otherwise isogenic with the wild-type strain 2.4.1, showed a normal growth rate at high light intensity compared with the wild type, with the levels of the B875 and reaction center spectral complexes being approximately 7% and 25%, respectively, of those found in the wild type. The deletion mutation correlated with a reduction in the size of the fixed photosynthetic unit from 15:1 in the wild type to 4:1 in the mutant. The level of the B800-850 complex was increased approximately twofold in the mutant strain. However, substantial amounts of the B875 and reaction center polypeptides were not incorporated into spectrally active complexes, suggesting the importance of other factors in the assembly of these complexes. Removal of the intercistronic stem-loops resulted in increased readthrough of the puf operon terminator to regions downstream, as well as altering the stability of the resulting puf operon-specific transcripts. A model is proposed which links ribosome stalling within the open reading frame K leader region of the puf operon transcript with chain termination.

Bacteria↗

Replacement of murine leukemia virus readthrough mechanism by human immunodeficiency virus frameshift allows synthesis of viral proteins and virus replication.

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model.

3T3 Cells↗

Inversion/dimerization of plasmids mediated by inverted repeats.

In contrast with earlier studies on the lambda and Escherichia coli genomes, recombination between inverted repeats on plasmids is highly efficient and shown to be recA-independent. In addition, the recombination product is exclusively a head-to-head inverted dimer. Here, we show that this recombination/rearrangement event can occur on different plasmid replicons and is not specific to the particular sequence within the inverted repeats. Transcription readthrough into the inverted repeats has little effect on this event. Genetic analysis has also indicated that most known recombination enzymes are not involved in this process. Specifically, single or double mutants defective in Holliday junction resolution systems (RuvABC and/or RecG/RusA) do not abolish this recombination/rearrangement event. This result does not support the previous models (i.e. the reciprocal-strand-switching and the cruciform-dumbbell models) in which intermediates containing Holliday junctions are proposed. Further analysis has demonstrated that the recombination/rearrangement frequency is dramatically (over 1000-fold) reduced if mismatches (2.8 %) are present within the inverted repeats. Mutations in dam, mutH and mutL genes partially or completely restored the recombination/rearrangement frequency to the level exhibited by the perfect inverted repeats, suggesting the formation of heteroduplexes during recombination/rearrangement. Sequencing analysis of the recombination/rearrangement products have indicated that the majority of the products do not involve crossing-over. We discuss a possible mechanism in which blockage of the lagging strand polymerase by a hairpin triggers recombination/rearrangement mediated by inverted repeats.

Base Sequence↗

The translational stop signal: codon with a context, or extended factor recognition element?

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, approximately 40,000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor 3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.

Base Sequence↗

Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis.

The insertion sequence IS6100 was shown to undergo intermolecular transposition from a temperature-sensitive delivery plasmid to the genome of the avermectin-producer Streptomyces avermitilis, creating cointegrates. Evidence from both Southern hybridization and the range of auxotrophic mutations present in a transposon library was consistent with random transposition. It was not possible to increase transposase expression by readthrough transcription from a copy of the tipA promoter located adjacent to the insertion sequence. This was in part due to the absence of a homologue of the Streptomyces lividans transcriptional activator TipAL in S. avermitilis. However, recombinant S. avermitilis strains carrying the S. lividans tip operon were also deficient for induction of the promoter. The frequency of reversion of different auxotrophic mutations by precise excision, involving recombination across 8 bp direct repeats, was shown to vary by at least five orders of magnitude. This dependence of recombination frequency on chromosomal location may contribute to the stability of repetitive modular type I polyketide biosynthetic genes.

Blotting, Southern↗

The complete nucleotide sequence and genome organization of red clover necrotic mosaic virus RNA-1.

The complete nucleotide sequence of red clover necrotic mosaic virus (RCNMV) RNA-1 has been determined. RNA-1 is 3889 nucleotides in length with a 5' terminal m7GpppA cap. The RNA contains three large open reading frames (ORFs): the 5' proximal ORF, encoding a 27-kDa polypeptide; the internal ORF, coding for a 57-kDa polypeptide; and the 3' terminal ORF, encoding the 37-kDa capsid protein. The sequence results confirm in vitro translation of 27-, 50-, and 37-kDa products but do not account for the observed 90-kDa product. A translational frameshift event from the 27- to the 57-kDa ORFs is proposed to explain the synthesis of the observed 90-kDa in vitro product. The putative translational frameshift region is structurally similar to several retrovirus frameshift regions and the putative barley yellow dwarf virus (BYDV) frameshift regions. Extensive amino acid homology was observed in the 57-kDa downstream ORF with the downstream domains of the carnation mottle virus (CarMV), turnip crinkle virus (TCV), maize chlorotic mottle virus (MCMV) readthrough, and BYDV fusion proteins. The 57-kDa ORF contained the conserved "GDD" motif. A significant alignment between the capsid proteins of RCNMV, CarMV, and TCV was also observed. Given the extensive amino acid sequence similarity of RCNMV, CarMV, and TCV polymerase and capsid proteins, we speculate that they are closely related, evolutionarily.

Amino Acid Sequence↗

Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones.

Promoters were assembled in nucleosomes or ligated to nucleosomes and transcribed with SP6 RNA polymerase or with mammalian RNA polymerase II and accessory factors. Neither polymerase would initiate transcription at a promoter in a nucleosome, but once engaged in transcription, both polymerases were capable of reading through a nucleosome. In the course of readthrough transcription, the histones were displaced from the DNA, as shown by the exposure of restriction sites and by a shift of the template to the position of naked DNA in a gel. It may be true, in general, that processive enzymes will traverse regions of DNA organized in nucleosomes and displace histones.

Animals↗

Molecular cloning and characterization of an interferon induced human cDNA with sequence homology to a mammalian peptide chain release factor.

Here we report the molecular cloning of several related human cDNAs from which a full-length sequence can be determined. The cDNAs encode a 2.8 kb mRNA that is strongly induced by interferon (IFN) gamma and the expression of which is not cell-restricted but observed in fibroblasts, macrophages and epithelial cells. The deduced amino acid sequence predicts a protein of 471 amino acids with high sequence similarity to a previously identified rabbit peptide chain release factor. Functional studies to demonstrate release factor activity showed that the protein encoded by this cDNA inhibited the readthrough activity of a yeast UGA suppressor tRNA in an in vitro translation system. The identification of this novel cDNA implies that translational control by IFN induced proteins may not be restricted to the initial steps of protein synthesis but may also act by regulation of peptide chain termination.

Amino Acid Sequence↗

The beet western yellows virus ST9-associated RNA shares structural and nucleotide sequence homology with carmo-like viruses.

Complementary DNAs corresponding to the virion RNAs of the ST9 isolate of beet western yellows virus (ST9 BWYV) were cloned and the complete, 2843 nucleotide sequence of a unique RNA associated with ST9 BWYV, the ST9-associated RNA (ST9 aRNA), was determined. Three ORFs with deduced translation products of molecular weights 22,600, 12,500, and 35,900, respectively, and a small ORF, potentially encoding for a 3400 molecular weight protein, were identified in the ST9 aRNA sequence. Possible gene products of the ST9 aRNA were identified by in vitro translation analysis of the ST9 BWYV virion RNAs (the ST9 BWYV genomic RNA [ST9 BWYV gRNA] and the ST9 aRNA) and of transcripts representing the nearly full-length ST9 aRNA, individual ORFs, and specific ORF combinations. Proteins of 23,000 and 28,000 M(r) were derived from ORF 1 transcripts, while proteins of 25,000 and 35,000 M(r) were derived from ORF 3 transcripts. Larger proteins of 85,000 and 45,000 M(r) were also identified, and shown to possibly arise by continuous translation of ORFs 1, 2, and 3 (by possible readthrough and frameshift), or ORFs 2 and 3 (by possible frameshift), respectively. No products were detected from transcripts representing only ORF 2. Computer database searches and analyses indicated that the ST9 aRNA shares significant nucleotide sequence homology with carmo-like viruses, but not with its helper virus, BWYV. Amino acid sequence analyses also indicated that the ST9 aRNA ORF 2 and ORF 3 regions share extensive homology with putative RNA-dependent RNA polymerases of carmo-like viruses.

Amino Acid Sequence↗

Transcriptional attenuation control of ermK, a macrolide-lincosamide-streptogramin B resistance determinant from Bacillus licheniformis.

ermK instructs bacteria to synthesize an erythromycin-inducible 23S rRNA methylase that confers resistance to the macrolide, lincosamide, and streptogramin B antibiotics. Expression of ermK is regulated by transcriptional attenuation, in contrast to other inducible erm genes, previously described, which are regulated translationally. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino-acid leader peptide together with its ribosome binding site. Additionally, the mRNA leader sequence can fold in either of two mutually exclusive conformations, one of which is postulated to form in the absence of induction and to contain two rho factor-independent terminators. Truncated transcription products ca. 210 and 333 nucleotides long were synthesized in the absence of induction, both in vivo and in vitro, as predicted by the transcriptional attenuation model; run-off transcription in vitro with rITP favored the synthesis of the full-length run-off transcript over that of the 210- and 333-nucleotide truncated products. Northern (RNA) blot analysis of transcripts synthesized in vivo in the absence of erythromycin indicated that transcription terminated at either of the two inverted complementary repeat sequences in the leader that were postulated to serve as rho factor-independent terminators; moreover, no full-length transcripts were detectable in the uninduced samples. In contrast, full-length (ca. 1,200-nucleotide) transcripts were only detected in RNA samples synthesized in vivo in the presence of erythromycin. Full-length transcripts formed in the absence of induction from transcriptional readthrough past the two proposed transcription terminators would fold in a way that would sequester the ribosome binding site together with the first two codons of the ErmK methylase, reducing its efficiency in translation. This feature could therefore provide additional control of expression in the absence of induction; however, such regulation, if operative, would act only secondarily, both in time and place, relative to transcriptional control. Analysis by reverse transcriptase mapping of in vivo transcripts from two primers that bracket the transcription terminator responsible for the 210-nucleotide truncated fragment supports the transcriptional attenuation model proposed and suggests further that the synthesis of the ermK message is initiated constitutively upstream of the proposed terminator but completed inductively downstream of this site.

Amino Acid Sequence↗

Polyamines enhance synthesis of the RNA polymerase sigma 38 subunit by suppression of an amber termination codon in the open reading frame.

The mechanisms by which polyamines stimulate synthesis of the RNA polymerase sigma(38) subunit in Escherichia coli were studied. Polyamine stimulation was observed only in strains in which the 33rd codon of RpoS mRNA is a UAG termination codon instead of a CAG codon for glutamine in wild-type E. coli. Readthrough of the termination codon by Gln-tRNA(supE) was stimulated by polyamines. This stimulation was found to be caused by an increase in both the level of suppressor tRNA(supE) and the binding affinity of Gln-tRNA(supE) for ribosomes. The stimulatory effect was observed with a UAG termination codon but not with UGA and UAA codons. Readthrough of the UAG termination codon at the 270th amino acid position of RpoS mRNA was also stimulated by polyamines, indicating that polyamines stimulate readthrough of a UAG codon regardless of its location within the RpoS mRNA. When cell viability of an E. coli strain having a termination codon in the 33rd position of RpoS mRNA was compared using cells cultured with or without putrescine, it was higher in cells cultured with putrescine than in cells cultured without putrescine. The level of sigma(38) subunit in the cells cultured with putrescine was higher than that in cells cultured without putrescine on days 2, 4, and 8, but the level of sigma(70) subunit was almost the same in cells cultured with or without putrescine. These results confirm that elevated expression of the rpoS gene is important for cell viability at late stationary phase.

Base Sequence↗

The virion-associated Gag-Pol is decreased in chimeric Moloney murine leukemia viruses in which the readthrough region is replaced by the frameshift region of the human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 translational frameshift event to synthesize the precursor of its enzymes, Gag-Pol, when ribosomes from the infected cells translate the full-length viral messenger RNA. Translation of the same RNA according to conventional translational rules produces Gag, the precursor of the structural proteins of the virus. The efficiency of the frameshift controls the ratio of Gag-Pol to Gag, which is critical for viral infectivity. The Moloney murine leukemia virus (MoMuLV) uses a different strategy, the programmed readthrough of a stop codon, to synthesize Gag-Pol. In this study, we investigated whether different forms of the HIV-1 frameshift region can functionally replace the readthrough signal in MoMuLV. Chimeric proviral DNAs were obtained by inserting into the MoMuLV genome the HIV-1 frameshift region encompassing the slippery sequence where the frameshift occurs, followed by the frameshift stimulatory signal. The inserted signal was either a simple stem-loop, previously considered as the stimulatory signal, or a longer bulged helix, now shown to be the complete stimulatory signal, or a mutated version of the complete signal with a three-nucleotide deletion. Although the three chimeric viruses can propagate essentially as the wild-type virus in NIH 3T3 cells, single-round infectivity assays revealed that the infectivity of the chimeric virions is about three to fivefold lower than that of the wild-type virions, depending upon the nature of the frameshift signal. It was also observed that the Gag-Pol to Gag ratio was decreased about two to threefold in chimeric virions. Comparison of the readthrough efficiency of MoMuLV to the HIV-1 frameshift efficiency, by monitoring the expression of a luciferase reporter in cultured cells, revealed that the frameshift efficiencies were only 30-60% of the readthrough efficiency. Altogether, these observations indicate that replacement of the readthrough region of MoMuLV with the frameshift region of HIV-1 results in virions that are replication competent, although less infectious than wild-type MoMuLV. This type of chimera could provide an interesting tool for in vivo studies of novel drugs targeted against the HIV-1 frameshift event.

Animals↗

Comparative study on genomes of two Japanese melon necrotic spot virus isolates.

Nucleotide sequences of the genomes of two Japanese Melon necrotic spot virus (MNSV) isolates, NH and NK were determined. The open reading frames (ORFs) in both genomes encode five proteins: p29 (the pre-readthrough domain of p89), p89 (the readthrough domain of p89 identified as the putative RNA-dependent RNA polymerase), p14 (the pre-readthrough domain of p7A), p7A (the putative movement protein), and p42 (coat protein, CP). Nucleotide and amino acid sequence identities of the five proteins of NH and NK isolates were estimated at 97.4-99.5% and 97.7-100%, respectively. NK isolate but not NH isolate infected systemically leaves of Cucumis melo plants. When deduced amino acid sequences of p7A proteins of NH and NK isolates were compared, only one difference at position 16 (serine in NH isolate and isoleucine in NK isolate) was observed. p7A protein is considered the putative movement protein. The serine of p7A protein of NH isolates may be involved in systemic infection. In addition, phylogenetic relationships of genes based on nucleotide sequences revealed that NH and NK isolates might form a group, and S isolate, serologically different from NH and NK isolates, might represent a distinct isolate not belonging to this group.

Amino Acid Sequence↗

The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae.

The complete sequences of the yeast a mating-type locus, MATa, and of the silent alpha cassette, HML alpha, have been determined. A segment of 642 nucleotides is unique to MATa, and a corresponding segment of 747 nucleotides is unique to MAT alpha. The major mRNAs (a1, a2, alpha 1 and alpha 2) encoded by MATa and MAT alpha have been aligned with the DNA sequence. The a1 mRNA is encoded entirely within the a-specific DNA sequence. The a2 mRNA, which is transcribed divergently from a1 mRNA, is encoded in a region common to both Mata and Mat alpha. The alpha 1 and alpha 2 mRNAs are also transcribed divergently and have their 5' starts about 240 nucleotides apart within the alpha-specific sequence. The amino acid sequences of the MAT proteins have been predicted from the DNA sequences. An unanticipated conclusion is that the a1 protein, containing 148 amino acids, results from readthrough of a UGA at codon 45. Polymorphic forms of the homologous outer segments of the HML alpha, MAT alpha, MATa and HMRa sequences suggest that the boundaries of the segments involved in mating-type switching are immediately adjacent to the a-specific and alpha-specific sequences.

Base Sequence↗

Transcriptional pausing, arrest, and readthrough at the adenovirus major late attenuation site.

RNA polymerase II (pol II) transcription complexes initiated from the adenovirus major late promoter can become blocked both in vitro and in vivo at a specific site within the first intron of the transcription unit. In vitro, polymerases that fail to read through the major late attenuation site remain stably bound to the template in a ternary complex that is indefinitely blocked from continuing elongation, a phenomenon referred to as "arrest." Elongation factor SII has been shown both to promote readthrough of this and other arrest sites and to stimulate a previously unknown 3' to 5' exonuclease activity of pol II. We have proposed that the two activities are related and that SII promotes readthrough by means of the enhancement of the exonuclease activity. In the experiments reported here, we have tested several features of that model. In particular, we have examined the hypothesis that SII stimulates readthrough by allowing the polymerase to undergo multiple cycles of removal and resynthesis of RNA bases preceding the attenuation site. In addition, we present experimental support for the proposal that the length of time polymerase pauses at the attenuation site is important to the efficiency of arrest. The results of these experiments are discussed in the context of the model.

Adenoviridae↗

Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II.

8-Oxoguanine (8-oxoG) is a major oxidative lesion produced in DNA by normal cellular metabolism or after exposure to exogenous sources such as ionizing radiation. Persistence of this lesion in DNA causes G to T transversions, with deleterious consequences for the cell. As a result, several repair processes have evolved to remove this lesion from the genome. It has been reported that 8-oxoG is subject to transcription-coupled repair (TCR), a process dedicated to removal of lesions from transcribed strands of expressed genes. A current model assumes that RNA polymerase arrest at the site of the lesion is required for initiation of TCR. As a first step to understand how TCR of 8-oxoG occurs, we have studied the effect of 8-oxoG on transcription elongation by T7 RNA polymerase (T7 RNAP) and rat liver RNA polymerase II (RNAPII). We have utilized an in vitro transcription system with purified RNA polymerase and initiation factors, and substrates containing a single 8-oxoG in the transcribed or in the non-transcribed strand downstream of the T7 promoter or the Adenovirus major late promoter. We found that 8-oxoG only slightly inhibited T7 RNAP transcription, with a readthrough frequency of up to 95%. Similarly, this lesion only transiently blocked transcription by RNAPII. However, changes in nucleotide concentration affected the extent of RNAPII blockage at the 8-oxoG. When this lesion was positioned in the non-transcribed strand, complete lesion bypass was observed with either polymerase. Binding of the Saccharomyces cerevisiae MSH2-MSH6 complex to 8-oxoG containing substrates did not increase the frequency of RNAPII arrest at the site of the lesion, suggesting that this complex was displaced by the elongating polymerase. These results are discussed in the context of possible models for TCR.

Animals↗

Isolation and characterization of four alternate c-erbB3 transcripts expressed in ovarian carcinoma-derived cell lines and normal human tissues.

ErbB-3 is a member of the epidermal growth factor receptor (EGFR/ErbB) family. In addition to the previously reported 6.2 kb full-length and 1.4 kb truncated c-erbB3 transcripts, we have observed a 1.7 kb c-erbB3 human transcript in Northern blots that specifically hybridizes to a probe of the extracellular domain of the receptor. Using 3'-RACE we have isolated four novel c-erbB3 cDNA clones of 1.6, 1.7, 2.1 and 2.3 kb from a human ovarian carcinoma-derived cell line. All four alternate transcripts are synthesized by readthrough of an intron and use of an alternative polyadenylation signal within this intron. Identical c-erbB3 transcripts are expressed in normal human placental tissues. Expression of these alternate transcripts is tissue-specific as indicated by Northern blot and RNase protection analyses. Fibroblasts transfected with expression vectors carrying these alternate c-erbB3 cDNA clones stably express truncated ErbB-3 products. Three of these four cDNA clones express a receptor product that is secreted. Immunoprecipitation analysis of primary cultures of human ovarian carcinomas also demonstrate the expression of a 90 kDa ErbB-3 related protein that is secreted. Furthermore, we demonstrate conservation of the exon-intron junctions between members of the erbB gene family in those regions of the gene encoding the extracellular domain. This gene structure is also conserved in the c-erbB1 homologues of Drosophila and C. elegans. Growth regulatory roles for related truncated ErbB products recently have been reported. It is, therefore, possible that the products of these four alternate c-erbB3 transcripts may also play important growth regulatory roles in normal and transformed cells.

Alternative Splicing↗

Enterococcus faecalis sex pheromone plasmid pAM373: analyses of TraA and evidence for its interaction with RpoB.

The Enterococcus faecalis plasmid pAM373 (36.7kb) encodes a mating response to the sex pheromone cAM373 secreted by recipient (plasmid-free) bacteria. Like certain other conjugative enterococcal plasmids, a key regulator of the pheromone response is a negatively acting protein, TraA, which is believed to interact with internalized pheromone to influence expression from a key transcriptional promoter P(0). An earlier report showed that in the case of pAM373 most, but not all, transposon-insertion mutations in traA differed from those in the case of pAD1 and pCF10 in that they did not give rise to the normally characteristic constitutive clumping. We show here that this phenomenon relates to a host effect involving an RpoB-related mutation associated with rifampin resistance. When harboring traA mutants, rifampin-sensitive hosts exhibited constitutive clumping, whereas rifampin-resistant hosts did not-despite the fact that the latter host exhibited a normal pheromone-inducible clumping response when harboring a wild-type plasmid. The data imply that TraA normally remains associated with the transcription complex after induction. In addition the promoter of traA, designated P(a), was shown to be located about 600bp upstream of the translational start site, as clones containing traA required this site to complement traA mutants in trans. Transcription from P(a) also gave rise to a short (130 nt) transcript, mD, expressed at a high level in uninduced cells. An earlier observation suggesting that TraA negatively affected transcriptional readthrough into the 3' end of traA from the t(ac) intrinsic bidirectional terminator between traA and the opposing, adjacent traC was supported by TraA complementation studies. Evidence is also presented suggesting that this regulation at t(ac) also involves an additional, possibly cis-acting, element.

Bacterial Proteins↗